Novel Substituted Bicyclic Aromatic Compounds as S-Nitrosoglutathione Reductase Inhibitors

ABSTRACT

The present invention is directed to novel substituted bicyclic aromatic compounds useful as S-nitrosoglutathione reductase (GSNOR) inhibitors, pharmaceutical compositions comprising such compounds, and methods of making and using the same.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/326,686, filed Jul. 9, 2014, which is a continuation of U.S.application Ser. No. 13/991,972, filed Jun. 6, 2013, issued as U.S. Pat.No. 8,785,643 on Jul. 22, 2014. U.S. application Ser. No. 13/991,972 isa 35 U.S.C. §371 national phase application of PCT/US2011/065490, filedDec. 16, 2011 (WO 2012/083165). PCT/US2011/065490 claims priority toU.S. Provisional Application Ser. No. 61/423,799, filed Dec. 16, 2010.Each of these applications is incorporated herein by reference in itsentirety for all purposes as if fully set forth herein.

FIELD OF THE INVENTION

The present invention is directed to novel bicyclic aromatic compounds,pharmaceutical compositions comprising such compounds, and methods ofmaking and using the same. These compounds are useful as inhibitors ofS-nitrosoglutathione reductase (GSNOR).

BACKGROUND

The chemical compound nitric oxide is a gas with chemical formula NO. NOis one of the few gaseous signaling molecules known in biologicalsystems, and plays an important role in controlling various biologicalevents. For example, the endothelium uses NO to signal surroundingsmooth muscle in the walls of arterioles to relax, resulting invasodilation and increased blood flow to hypoxic tissues. NO is alsoinvolved in regulating smooth muscle proliferation, platelet function,and neurotransmission, and plays a role in host defense. Although NO ishighly reactive and has a lifetime of a few seconds, it can both diffusefreely across membranes and bind to many molecular targets. Theseattributes make NO an ideal signaling molecule capable of controllingbiological events between adjacent cells and within cells.

NO is a free radical gas, which makes it reactive and unstable, thus NOis short lived in vivo, having a half life of 3-5 seconds underphysiologic conditions. In the presence of oxygen, NO can combine withthiols to generate a biologically important class of stable NO adductscalled S-nitrosothiols (SNO's). This stable pool of NO has beenpostulated to act as a source of bioactive NO and as such appears to becritically important in health and disease, given the centrality of NOin cellular homeostasis (Stamler et al., Proc. Natl. Acad. Sci. USA,89:7674-7677 (1992)). Protein SNO's play broad roles in the function ofcardiovascular, respiratory, metabolic, gastrointestinal, immune, andcentral nervous system (Foster et al., Trends in Molecular Medicine, 9(4):160-168, (2003)). One of the most studied SNO's in biologicalsystems is S-nitrosoglutathione (GSNO) (Gaston et al., Proc. Natl. Acad.Sci. USA 90:10957-10961 (1993)), an emerging key regulator in NOsignaling since it is an efficient trans-nitrosating agent and appearsto maintain an equilibrium with other S-nitrosated proteins (Liu et al.,Nature, 410:490-494 (2001)) within cells. Given this pivotal position inthe NO—SNO continuum, GSNO provides a therapeutically promising targetto consider when NO modulation is pharmacologically warranted.

In light of this understanding of GSNO as a key regulator of NOhomeostasis and cellular SNO levels, studies have focused on examiningendogenous production of GSNO and SNO proteins, which occurs downstreamfrom the production of the NO radical by the nitric oxide synthetase(NOS) enzymes. More recently there has been an increasing understandingof enzymatic catabolism of GSNO which has an important role in governingavailable concentrations of GSNO and consequently available NO andSNO's.

Central to this understanding of GSNO catabolism, researchers haverecently identified a highly conserved S-nitrosoglutathione reductase(GSNOR) (Jensen et al., Biochem J., 331:659-668 (1998); Liu et al.,(2001)). GSNOR is also known as glutathione-dependent formaldehydedehydrogenase (GSH-FDH), alcohol dehydrogenase 3 (ADH-3) (Uotila andKoivusalo, Coenzymes and Cofactors., D. Dolphin, ed. pp. 517-551 (NewYork, John Wiley & Sons, (1989)), and alcohol dehydrogenase 5 (ADH-5).Importantly GSNOR shows greater activity toward GSNO than othersubstrates (Jensen et al., (1998); Liu et al., (2001)) and appears tomediate important protein and peptide denitrosating activity inbacteria, plants, and animals. GSNOR appears to be the majorGSNO-metabolizing enzyme in eukaryotes (Liu et al., (2001)). Thus, GSNOcan accumulate in biological compartments where GSNOR activity is low orabsent (e.g., airway lining fluid) (Gaston et al., (1993)).

Yeast deficient in GSNOR accumulate S-nitrosylated proteins which arenot substrates of the enzyme, which is strongly suggestive that GSNOexists in equilibrium with SNO-proteins (Liu et al., (2001)). Preciseenzymatic control over ambient levels of GSNO and thus SNO-proteinsraises the possibility that GSNO/GSNOR may play roles across a host ofphysiological and pathological functions including protection againstnitrosative stress wherein NO is produced in excess of physiologicneeds. Indeed, GSNO specifically has been implicated in physiologicprocesses ranging from the drive to breathe (Lipton et al., Nature,413:171-174 (2001)) to regulation of the cystic fibrosis transmembraneregulator (Zaman et al., Biochem Biophys Res Commun, 284:65-70 (2001)),to regulation of vascular tone, thrombosis, and platelet function (deBelder et al., Cardiovasc Res.; 28(5):691-4 (1994)), Z. Kaposzta, etal., Circulation; 106(24): 3057-3062, (2002)) as well as host defense(de Jesus-Berrios et al., Curr. Biol., 13:1963-1968 (2003)). Otherstudies have found that GSNOR protects yeast cells against nitrosativestress both in vitro (Liu et al., (2001)) and in vivo (de Jesus-Berrioset al., (2003)).

Collectively, data suggest GSNO as a primary physiological ligand forthe enzyme S-nitrosoglutathione reductase (GSNOR), which catabolizesGSNO and consequently reduces available SNO's and NO in biologicalsystems (Liu et al., (2001)), (Liu et al., Cell, 116(4), 617-628(2004)), and (Que et al., Science, 308, (5728):1618-1621 (2005)). Assuch, this enzyme plays a central role in regulating local and systemicbioactive NO. Since perturbations in NO bioavailability has been linkedto the pathogenesis of numerous disease states, including hypertension,atherosclerosis, thrombosis, asthma, gastrointestinal disorders,inflammation, and cancer, agents that regulate GSNOR activity arecandidate therapeutic agents for treating diseases associated with NOimbalance.

Nitric oxide (NO), S-nitrosoglutathione (GSNO), and S-nitrosoglutathionereductase (GSNOR) regulate normal lung physiology and contribute to lungpathophysiology. Under normal conditions, NO and GSNO maintain normallung physiology and function via their anti-inflammatory andbronchodilatory actions. Lowered levels of these mediators in pulmonarydiseases such as asthma, chronic obstructive pulmonary disease (COPD)may occur via up-regulation of GSNOR enzyme activity. These loweredlevels of NO and GSNO, and thus lowered anti-inflammatory capabilities,are key events that contribute to pulmonary diseases and which canpotentially be reversed via GSNOR inhibition.

S-nitrosoglutathione (GSNO) has been shown to promote repair and/orregeneration of mammalian organs, such as the heart (Lima et al., 2010),blood vessels (Lima et al., 2010) skin (Georgii et al., 2010), eye orocular structures (Haq et al., 2007) and liver (Prince et al., 2010).S-nitrosoglutathione reductase (GSNOR) is the major catabolic enzyme ofGSNO. Inhibition of GSNOR is thought to increase endogenous GSNO.

Inflammatory bowel diseases (IBD's), including Crohn's and ulcerativecolitis, are chronic inflammatory disorders of the gastrointestinal (GI)tract, in which NO, GSNO, and GSNOR can exert influences. Under normalconditions, NO and GSNO function to maintain normal intestinalphysiology via anti-inflammatory actions and maintenance of theintestinal epithelial cell barrier. In IBD, reduced levels of GSNO andNO are evident and likely occur via up-regulation of GSNOR activity. Thelowered levels of these mediators contribute to the pathophysiology ofIBD via disruption of the epithelial barrier via dysregulation ofproteins involved in maintaining epithelial tight junctions. Thisepithelial barrier dysfunction, with the ensuing entry ofmicro-organisms from the lumen, and the overall loweredanti-inflammatory capabilities in the presence of lowered NO and GSNO,are key events in IBD progression that can be potentially influenced bytargeting GSNOR.

Cell death is the crucial event leading to clinical manifestation ofhepatotoxicity from drugs, viruses and alcohol. Glutathione (GSH) is themost abundant redox molecule in cells and thus the most importantdeterminant of cellular redox status. Thiols in proteins undergo a widerange of reversible redox modifications during times of exposure toreactive oxygen and reactive nitrogen species, which can affect proteinactivity. The maintenance of hepatic GSH is a dynamic process achievedby a balance between rates of GSH synthesis, GSH and GSSG efflux, GSHreactions with reactive oxygen species and reactive nitrogen species andutilization by GSH peroxidase. Both GSNO and GSNOR play roles in theregulation of protein redox status by GSH.

Acetaminophen overdoses are the leading cause of acute liver failure(ALF) in the United States, Great Britain and most of Europe. More than100,000 calls to the U.S. Poison Control Centers, 56,000 emergency roomvisits, 2600 hospitalizations, nearly 500 deaths are attributed toacetaminophen in this country annually. Approximately, 60% recoverwithout needing a liver transplant, 9% are transplanted and 30% ofpatients succumb to the illness. The acetaminophen-related death rateexceeds by at least three-fold the number of deaths due to all otheridiosyncratic drug reactions combined (Lee, Hepatol Res 2008; 38 (Suppl.1):S3-S8).

Liver transplantation has become the primary treatment for patients withfulminant hepatic failure and end-stage chronic liver disease, as wellas certain metabolic liver diseases. Thus, the demand fortransplantation now greatly exceeds the availability of donor organs. Ithas been estimated that more than 18 000 patients are currentlyregistered with the United Network for Organ Sharing (UNOS) and that anadditional 9000 patients are added to the liver transplant waiting listeach year, yet less than 5000 cadaveric donors are available fortransplantation.

Currently, there is a great need in the art for diagnostics,prophylaxis, ameliorations, and treatments for medical conditionsrelating to increased NO synthesis and/or increased NO bioactivity. Inaddition, there is a significant need for novel compounds, compositions,and methods for preventing, ameliorating, or reversing otherNO-associated disorders. The present invention satisfies these needs.

SUMMARY

The present invention provides novel bicyclic aromatic compounds. Thesecompounds are useful as S-nitrosoglutathione reductase (“GSNOR”)inhibitors. The invention encompasses pharmaceutically acceptable salts,stereoisomers, prodrugs, metabolites, and N-oxides of the describedcompounds. Also encompassed by the invention are pharmaceuticalcompositions comprising at least one compound of the invention and atleast one pharmaceutically acceptable carrier.

The compositions of the present invention can be prepared in anysuitable pharmaceutically acceptable dosage form.

The present invention provides a method for inhibiting GSNOR in asubject in need thereof. Such a method comprises administering atherapeutically effective amount of a pharmaceutical compositioncomprising at least one GSNOR inhibitor or a pharmaceutically acceptablesalt, stereoisomer, prodrug, metabolite or N-oxide thereof, incombination with at least one pharmaceutically acceptable carrier. TheGSNOR inhibitor can be a novel compound according to the invention, orit can be a known compound which previously was not known to be aninhibitor of GSNOR.

The present invention also provides a method of treating a disorderameliorated by NO donor therapy in a subject in need thereof. Such amethod comprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt, stereoisomer, prodrug, metabolite, orN-oxide thereof, in combination with at least one pharmaceuticallyacceptable carrier. The GSNOR inhibitor can be a novel compoundaccording to the invention, or it can be a known compound whichpreviously was not known to be an inhibitor of GSNOR.

The present invention also provides a method of treating a cellproliferative disorder in a subject in need thereof. Such a methodcomprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt, stereoisomer, prodrug, metabolite, orN-oxide thereof, in combination with at least one pharmaceuticallyacceptable carrier. The GSNOR inhibitor can be a novel compoundaccording to the invention, or it can be a known compound whichpreviously was not known to be an inhibitor of GSNOR.

The methods of the invention encompass administration with one or moresecondary active agents. Such administration can be sequential or in acombination composition.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publiclyavailable publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control.

Both the foregoing summary and the following detailed description areexemplary and explanatory and are intended to provide further details ofthe compositions and methods as claimed. Other objects, advantages, andnovel features will be readily apparent to those skilled in the art fromthe following detailed description.

DETAILED DESCRIPTION A. Overview of the Invention

Until recently, S-nitrosoglutathione reductase (GSNOR) was known tooxidize the formaldehyde glutathione adduct, S-hydroxymethylglutathione.GSNOR has since been identified in a variety of bacteria, yeasts,plants, and animals and is well conserved. The proteins from E. coli, S.cerevisiae and mouse macrophages share over 60% amino acid sequenceidentity. GSNOR activity (i.e., decomposition of GSNO when NADH ispresent as a required cofactor) has been detected in E. coli, in mousemacrophages, in mouse endothelial cells, in mouse smooth muscle cells,in yeasts, and in human HeLa, epithelial, and monocyte cells. HumanGSNOR nucleotide and amino acid sequence information can be obtainedfrom the National Center for Biotechnology Information (NCBI) databasesunder Accession Nos. M29872, NM_(—)000671. Mouse GSNOR nucleotide andamino acid sequence information can be obtained from NCBI databasesunder Accession Nos. NM_(—)007410. In the nucleotide sequence, the startsite and stop site are underlined. CDS designates coding sequence. SNPdesignates single nucleotide polymorphism. Other related GSNORnucleotide and amino acid sequences, including those of other species,can be found in U.S. Patent Application 2005/0014697.

In accord with the present invention, GSNOR has been shown to functionin vivo and in vitro to metabolize S-nitrosoglutathione (GSNO) andprotein S-nitrosothiols (SNOs) to modulate NO bioactivity, bycontrolling the intracellular levels of low mass NO donor compounds andpreventing protein nitrosylation from reaching toxic levels.

Based on this, it follows that inhibition of this enzyme potentiatesbioactivity in diseases in which NO donor therapy is indicated, inhibitsthe proliferation of pathologically proliferating cells, and increasesNO bioactivity in diseases where this is beneficial.

The present invention provides pharmaceutical agents that are potentinhibitors of GSNOR. In particular, provided are substituted bicyclicaromatic analogs having the structures depicted below (Formula I), or apharmaceutically acceptable salt, stereoisomer, prodrug, metabolite, orN-oxide thereof.

whereinZ₁ is selected from the group consisting of CR_(2a) and N;Z₂ is selected from the group consisting of CR_(2b) and N;Z₃ is selected from the group consisting of CR_(2c) and N;with the proviso that at least 1 of Z₁, Z₂, or Z₃ must be N;m is selected from the group consisting of 0, 1, 2, or 3;R₁ is independently selected from the group consisting of chloro,fluoro, and bromo;R_(2a), R_(2b), and R_(2c), are independently selected from the groupconsisting of hydrogen, halogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl,cyano, C₁-C₃ alkoxy, and N(CH₃)₂;X is selected from the group consisting of

n is selected from 0, 1, and 2;R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members;A is selected from the group consisting of

As used in this context, the term “analog” refers to a compound havingsimilar chemical structure and function as compounds of Formula I thatretains the bicyclic aromatic ring.

Some bicyclic aromatic analogs of the invention can also exist invarious isomeric forms, including configurational, geometric, andconformational isomers, as well as existing in various tautomeric forms,particularly those that differ in the point of attachment of a hydrogenatom. As used herein, the term “isomer” is intended to encompass allisomeric forms of a compound including tautomeric forms of the compound.

Illustrative compounds having asymmetric centers can exist in differentenantiomeric and diastereomeric forms. A compound can exist in the formof an optical isomer or a diastereomer. Accordingly, the inventionencompasses compounds in the forms of their optical isomers,diastereomers and mixtures thereof, including racemic mixtures.

It should be noted that if there is a discrepancy between a depictedstructure and a name given to that structure, the depicted structurecontrols. In addition, if the stereochemistry of a structure or aportion of a structure is not indicated with, for example, bold, wedged,or dashed lines, the structure or portion of the structure is to beinterpreted as encompassing all stereoisomers of the described compound.

B. S-Nitrosoglutathione Reductase Inhibitors 1. Inventive Compounds

In one of its aspects the present invention provides compounds havingthe structure shown in Formula I, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

whereinZ₁ is selected from the group consisting of CR_(2a) and N;Z₂ is selected from the group consisting of CR_(2b) and N;Z₃ is selected from the group consisting of CR_(2c) and N;with the proviso that at least 1 of Z₁, Z₂, or Z₃ must be N;m is selected from the group consisting of 0, 1, 2, or 3;R₁ is independently selected from the group consisting of chloro,fluoro, and bromo;R_(2a), R_(2b), and R_(2c), are independently selected from the groupconsisting of hydrogen, halogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl,cyano, C₁-C₃ alkoxy, and N(CH₃)₂;X is selected from the group consisting of

n is selected from 0, 1, and 2;R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members; andA is selected from the group consisting of

In a further aspect of the invention wherein when Z₁ and Z₂ are both N,and Z₃ is CR_(2c), then R_(2c) is selected from the group consisting ofhydrogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl, and cyano.

In a further aspect of the invention, m is selected from the groupconsisting of 0 and 1; R_(2a), R_(2b), and R_(2c), are independentlyselected from the group consisting of hydrogen, chloro, fluoro, methyl,trifluoromethyl, cyano, methoxy, and N(CH₃)₂; n is selected from thegroup consisting of 0 and 1; and R₃ is independently selected from thegroup consisting of fluoro, chloro, methyl, trifluoromethyl, cyano,methoxy, and NR₄R_(4′) where R₄ and R_(4′) are methyl, or alternativelytogether with the said N form the ring aziridin-1-yl or morpholino.

In a further aspect of the invention, X is

In a further aspect of the invention, A is COOH.

In one of its aspects the present invention provides a compound having astructure shown in Formula II, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

whereinZ₂ is selected from the group consisting of CR_(2b) and N;Z₃ is selected from the group consisting of CR_(2c) and N;m is selected from the group consisting of 0, 1, 2, or 3;R₁ is independently selected from the group consisting of chloro,fluoro, and bromo;R_(2b) and R_(2c) are independently selected from the group consistingof hydrogen, halogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃alkoxy, and N(CH₃)₂;X is selected from the group consisting of

n is selected from 0, 1, and 2;R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members; andA is selected from the group consisting of

In a further aspect of the invention, when Z₂ of Formula II is N and Z₃is CR_(2c), then R_(2c) is selected from the group consisting ofhydrogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl, and cyano.

In a further aspect of the invention, m of Formula II is selected fromthe group consisting of 0 and 1; R_(2a), R_(2b), and R_(2c), areindependently selected from the group consisting of hydrogen, chloro,fluoro, methyl, trifluoromethyl, cyano, methoxy, and N(CH₃)₂; n isselected from the group consisting of 0 and 1; and R₃ is independentlyselected from the group consisting of fluoro, chloro, methyl,trifluoromethyl, cyano, methoxy, and NR₄R_(4′) where R₄ and R_(4′) aremethyl, or alternatively together with the said N form the ringaziridin-1-yl or morpholino.

In one of its aspects the present invention provides a compound having astructure shown in Formula III, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

whereinZ₂ is selected from the group consisting of CR_(2b) and N;Z₃ is selected from the group consisting of CR_(2c) and N;with the proviso that at least 1 of Z₂ or Z₃ must be N;m is selected from the group consisting of 0, 1, 2, or 3;R₁ is independently selected from the group consisting of chloro,fluoro, and bromo;R_(2a), R_(2b), and R_(2c), are independently selected from the groupconsisting of hydrogen, halogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl,cyano, C₁-C₃ alkoxy, and N(CH₃)₂;X is selected from the group consisting of

n is selected from 0, 1, and 2;R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members; andA is selected from the group consisting of

In one of its aspects the present invention provides a compound having astructure shown in Formula IV, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

whereinm is selected from the group consisting of 0, 1, 2, or 3;R₁ is independently selected from the group consisting of chloro,fluoro, and bromo;R_(2b) and R_(2c) are independently selected from the group consistingof hydrogen, halogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃alkoxy, and N(CH₃)₂;X is selected from the group consisting of

n is selected from 0, 1, and 2;R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members; andA is selected from the group consisting of

In a further aspect of the invention, m of Formula IV is selected fromthe group consisting of 0 and 1; R_(2a), R_(2b), and R_(2c), areindependently selected from the group consisting of hydrogen, chloro,fluoro, methyl, trifluoromethyl, cyano, methoxy, and N(CH₃)₂;

n is selected from the group consisting of 0 and 1; and R₃ isindependently selected from the group consisting of fluoro, chloro,methyl, trifluoromethyl, cyano, methoxy, and NR₄R_(4′) where R₄ andR_(4′) are methyl, or alternatively together with the said N form thering aziridin-1-yl or morpholino.

In a further aspect of the invention, X of Formula IV is

In a further aspect of the invention, A of Formula IV is COOH.

In a further aspect of the invention, suitable compounds of Formula IVinclude, but are not limited to:

-   4-(6-hydroxy-3-methylquinolin-2-yl)benzoic acid;-   2-(4-(1H-tetrazol-5-yl)phenyl)-3-methylquinolin-6-ol;-   4-(6-hydroxyquinolin-2-yl)benzoic acid;-   2-(4-(1H-tetrazol-5-yl)phenyl)quinolin-6-ol;-   1-(6-hydroxyquinolin-2-yl)piperidine-4-carboxylic acid;-   (1r,4r)-4-(6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;-   (1s,4s)-4-(6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;-   3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid;-   2-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid;-   2-fluoro-4-(6-hydroxyquinolin-2-yl)benzoic acid;-   2-(4-(2H-tetrazol-5-yl)phenyl)-4-chloroquinolin-6-ol;-   3-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-5(2H)-one;-   3-fluoro-4-(6-hydroxyquinolin-2-yl)benzoic acid;-   4-(6-hydroxyquinolin-2-yl)-3-methoxybenzoic acid;-   5-(6-hydroxyquinolin-2-yl)thiophene-2-carboxylic acid;-   4-(6-hydroxyquinolin-2-yl)cyclohex-3-enecarboxylic acid;-   4-(3-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;-   4-(4-chloro-3-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;-   4-(3-chloro-6-hydroxyquinolin-2-yl)benzoic acid;-   3-(2-fluoro-4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-5(4H)-one;-   3-(3-fluoro-4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-5(4H)-one;-   4-(4-chloro-6-hydroxyquinolin-2-yl)benzoic acid;-   2-(2-chloro-4-(2H-tetrazol-5-yl)phenyl)quinolin-6-ol;-   5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,3,4-oxadiazol-2(3H)-one;-   3-(dimethylamino)-4-(6-hydroxyquinolin-2-yl)benzoic acid;-   4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;-   4-(6-hydroxyquinolin-2-yl)-3-methylbenzoic acid;-   4-(3-chloro-6-hydroxyquinolin-2-yl)-3-fluorobenzoic acid;-   3-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-thiadiazol-5(2H)-one;-   4-(6-hydroxyquinolin-2-yl)-3-(trifluoromethyl)benzoic acid;-   4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)benzoic acid;-   2-(4-carboxyphenyl)-6-hydroxyquinoline 1-oxide;-   5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,3,4-thiadiazol-2(3H)-one;-   5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-3(2H)-one;-   (1r,4r)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic    acid;-   (1s,4s)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic    acid;-   3-chloro-4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;-   2-(5-(2H-tetrazol-5-yl)thiophen-2-yl)quinolin-6-ol;-   5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-thiadiazol-3(2H)-one;-   3-fluoro-4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;-   1-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)piperidine-4-carboxylic    acid;-   4-(5-chloro-6-hydroxyquinolin-2-yl)benzoic acid;-   (1r,4r)-4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylic    acid;-   (1s,4s)-4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylic    acid;-   3-chloro-4-(5-fluoro-6-hydroxyquinolin-2-yl)benzoic acid; and-   4-(5-fluoro-6-hydroxyquinolin-2-yl)-3-methylbenzoic acid.

In one of its aspects the present invention provides a compound having astructure shown in Formula V, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

whereinm is selected from the group consisting of 0, 1, 2, or 3;R₁ is independently selected from the group consisting of chloro,fluoro, and bromo;R₂ is selected from the group consisting of hydrogen, halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and N(CH₃)₂;X is selected from the group consisting of

n is selected from 0, 1, and 2;R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members; andA is selected from the group consisting of

In a further aspect of the invention, R_(2c) is selected from the groupconsisting of hydrogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl, and cyano.

In a further aspect of the invention, suitable compounds of Formula Vinclude, but are not limited to:

-   4-(6-hydroxyquinazolin-2-yl)benzoic acid;-   4-(6-hydroxy-4-methylquinazolin-2-yl)benzoic acid;-   3-chloro-4-(6-hydroxyquinazolin-2-yl)benzoic acid;-   4-(6-hydroxyquinazolin-2-yl)-3-methylbenzoic acid;-   4-(6-hydroxyquinazolin-2-yl)-3-methoxybenzoic acid;-   3-fluoro-4-(6-hydroxyquinazolin-2-yl)benzoic acid; and-   1-(6-hydroxyquinazolin-2-yl)piperidine-4-carboxylic acid.

In one of its aspects the present invention provides a compound having astructure shown in Formula VI, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

whereinm is selected from the group consisting of 0, 1, 2, or 3;R₁ is independently selected from the group consisting of chloro,fluoro, and bromo;R_(2b) is selected from the group consisting of hydrogen, halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and N(CH₃)₂;X is selected from the group consisting of

n is selected from 0, 1, and 2;R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members; andA is selected from the group consisting of

In a further aspect of the invention, suitable compounds of Formula VIinclude, but are not limited to:

-   4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)benzoic acid;-   2-(4-(2H-tetrazol-5-yl)phenyl)-3-(trifluoromethyl)quinoxalin-6-ol;-   1-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)piperidine-4-carboxylic    acid;-   4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)cyclohex-3-enecarboxylic    acid;-   3-chloro-4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)benzoic    acid;-   4-(6-hydroxyquinoxalin-2-yl)benzoic acid;-   3-fluoro-4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)benzoic    acid;-   (1r,4r)-4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)cyclohexanecarboxylic    acid; and-   (1s,4s)-4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)cyclohexanecarboxylic    acid.

In one of its aspects the present invention provides a compound having astructure shown in Formula VII, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

whereinm is selected from the group consisting of 0, 1, 2, or 3;R₁ is independently selected from the group consisting of chloro,fluoro, and bromo;X is selected from the group consisting of

n is selected from 0, 1, and 2;R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members; andA is selected from the group consisting of

In a further aspect of the invention, suitable compounds of Formula VIIinclude, but are not limited to:

-   3-(4-carboxyphenyl)-7-hydroxybenzo[e][1,2,4]triazine 1-oxide;-   4-(7-hydroxybenzo[e][1,2,4]triazin-3-yl)benzoic acid;-   3-(4-carboxy-2-fluorophenyl)-7-hydroxybenzo[e][1,2,4]triazine    1-oxide; and-   3-fluoro-4-(7-hydroxybenzo[e][1,2,4]triazin-3-yl)benzoic acid.

In one of its aspects the present invention provides a compound having astructure shown in Formula VIII, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

whereinm is selected from the group consisting of 0, 1, 2, or 3;R₁ is independently selected from the group consisting of chloro,fluoro, and bromo;R_(2a) and R_(2c) are independently selected from the group consistingof hydrogen, halogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃alkoxy, and N(CH₃)₂;X is selected from the group consisting of

n is selected from 0, 1, and 2;R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members; andA is selected from the group consisting of

In a further aspect of the invention, a suitable compound of FormulaVIII includes, but is not limited to:

-   4-(7-hydroxyisoquinolin-3-yl)benzoic acid;-   3-fluoro-4-(7-hydroxyisoquinolin-3-yl)benzoic acid; and-   4-(7-hydroxy-4-methylisoquinolin-3-yl)benzoic acid.

In one of its aspects the present invention provides a compound having astructure shown in Formula IX, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

whereinm is selected from the group consisting of 0, 1, 2, or 3;R₁ is independently selected from the group consisting of chloro,fluoro, and bromo;R_(2a) and R_(2b) are independently selected from the group consistingof hydrogen, halogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃alkoxy, and N(CH₃)₂;X is selected from the group consisting of

n is selected from 0, 1, and 2;R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinatedC₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) where R₄ and R_(4′) areindependently selected from the group consisting of C₁-C₃ alkyl, or R₄when taken together with R_(4′) form a ring with 3 to 6 members; andA is selected from the group consisting of

In a further aspect of the invention, suitable compounds of Formula IXinclude, but are not limited to:

-   4-(7-hydroxyquinolin-3-yl)benzoic acid; and-   3-(4-carboxyphenyl)-7-hydroxyquinoline 1-oxide.

In one of its aspects the present invention provides a compound having astructure shown in Formula X, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

whereinm is selected from the group consisting of 0, 1, 2, or 3;R₁ is independently selected from the group consisting of chloro,fluoro, and bromo;R_(2a) is selected from the group consisting of hydrogen, halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and N(CH₃)₂;X is selected from the group consisting of

n is selected from 0, 1, and 2;R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members;A is selected from the group consisting of

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

The compounds described herein may have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom maybe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Many geometric isomers of olefins, C═N double bonds, and the like canalso be present in the compounds described herein, and all such stableisomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral, diastereomeric, racemic, and geometricisomeric forms of a structure are intended, unless the specificstereochemistry or isomeric form is specifically indicated. Alltautomers of shown or described compounds are also considered to be partof the present invention.

It is to be understood that isomers arising from such asymmetry (e.g.,all enantiomers and diastereomers) are included within the scope of theinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof. Alkenes can include either the E- or Z-geometry,where appropriate.

2. Representative Compounds

Examples 1-71 list representative novel substituted bicyclic aromaticanalogs of the invention. The synthetic methods that can be used toprepare each compound are detailed in Examples 1-71, with reference tothe synthetic schemes depicted before Example 1, and reference tointermediates described in Example 72. Supporting mass spectrometry dataand/or proton NMR data for each compound is also included in Examples1-71. GSNOR inhibitor activity was determined by the assay described inExample 73 and IC₅₀ values were obtained. GSNOR inhibitor compounds inExamples 1-71 had an IC₅₀ of about <10 μM. GSNOR inhibitor compounds inExamples 1-4, 6, 8, 10-14, 16-35, 37-43, 45, 47-50, 52-62, 65-68, 70-71had an IC₅₀ of about <0.5 μM. GSNOR inhibitor compounds in Examples 1-4,8, 10-14, 17-28, 30, 31, 37, 40-41, 43, 45, 47-50, 52-58, 60-61, 65,67-68, and 70-71 had an IC₅₀ of about <0.1 μM.

C. Definitions

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill in the art given the context in which it is used, “about”will mean up to plus or minus 10% of the particular term.

The term “acyl” includes compounds and moieties that contain the acetylradical (CH₃CO—) or a carbonyl group to which a straight or branchedchain lower alkyl residue is attached.

The term “alkyl” as used herein refers to a straight or branched chain,saturated hydrocarbon having the indicated number of carbon atoms. Forexample, (C₁-C₆) alkyl is meant to include, but is not limited tomethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl, hexyl, isohexyl, and neohexyl. An alkyl group canbe unsubstituted or optionally substituted with one or more substituentsas described herein.

The term “alkenyl” as used herein refers to a straight or branched chainunsaturated hydrocarbon having the indicated number of carbon atoms andat least one double bond. Examples of a (C₂-C₈) alkenyl group include,but are not limited to, ethylene, propylene, 1-butylene, 2-butylene,isobutylene, sec-butylene, 1-pentene, 2-pentene, isopentene, 1-hexene,2-hexene, 3-hexene, isohexene, 1-heptene, 2-heptene, 3-heptene,isoheptene, 1-octene, 2-octene, 3-octene, 4-octene, and isooctene. Analkenyl group can be unsubstituted or optionally substituted with one ormore substituents as described herein.

The term “alkynyl” as used herein refers to a straight or branched chainunsaturated hydrocarbon having the indicated number of carbon atoms andat least one triple bond. Examples of a (C₂-C₈) alkynyl group include,but are not limited to, acetylene, propyne, 1-butyne, 2-butyne,1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne,2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne, and 4-octyne. Analkynyl group can be unsubstituted or optionally substituted with one ormore substituents as described herein.

The term “alkoxy” as used herein refers to an —O-alkyl group having theindicated number of carbon atoms. For example, a (C₁-C₆) alkoxy groupincludes —O-methyl, —O-ethyl, —O-propyl, —O-isopropyl, —O-butyl,—O-sec-butyl, —O-tert-butyl, —O-pentyl, —O— isopentyl, —O-neopentyl,—O-hexyl, —O-isohexyl, and —O-neohexyl.

The term “aminoalkyl” as used herein, refers to an alkyl group(typically one to six carbon atoms) wherein one or more of the C₁-C₆alkyl group's hydrogen atoms is replaced with an amine of formula—N(R^(c))₂, wherein each occurrence of R^(c) is independently —H or(C₁-C₆) alkyl. Examples of aminoalkyl groups include, but are notlimited to, —CH₂NH₂, —CH₂CH₂NH₂, —CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂CH₂NH₂,—CH₂CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂N(CH₃)₂,t-butylaminomethyl, isopropylaminomethyl, and the like.

The term “aryl” as used herein refers to a 5- to 14-membered monocyclic,bicyclic, or tricyclic aromatic ring system. Examples of an aryl groupinclude phenyl and naphthyl. An aryl group can be unsubstituted oroptionally substituted with one or more substituents as described hereinbelow. Examples of aryl groups include phenyl or aryl heterocycles suchas, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole,triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine,pyridazine, and pyrimidine, and the like.

As used herein, the term “bioactivity” indicates an effect on one ormore cellular or extracellular process (e.g., via binding, signaling,etc.) which can impact physiological or pathophysiological processes.

The term “carbonyl” includes compounds and moieties which contain acarbon connected with a double bond to an oxygen atom. Examples ofmoieties containing a carbonyl include, but are not limited to,aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “carboxy” or “carboxyl” means a —COOH group or carboxylic acid.

“Acidic moiety” as used herein is defined as a carboxylic acid or acarboxylic acid bioisostere. Bioisosteres are substituents or groupswith similar physical or chemical properties which produce broadlysimilar biological properties to a chemical compound. For a review ofbioisosteres, see J. Med. Chem, 2011, 54, 2529-2591. Examples of “acidicmoiety” include but are not limited to

“Pharmacophore” is defined as “a set of structural features in amolecule that is recognized at a receptor site and is responsible forthat molecule's biological activity” (Gund, Prog. Mol. Subcell. Biol.,5: pp 117-143 (1977)).

The term “C_(m)-C_(n)” means “m” number of carbon atoms to “n” number ofcarbon atoms. For example, the term “C₁-C₆” means one to six carbonatoms (C₁, C₂, C₃, C₄, C₅, or C₆). The term “C₂-C₆” includes two to sixcarbon atoms (C₂, C₃, C₄, C₅, or C₆). The term “C₃-C₆” includes three tosix carbon atoms (C₃, C₄, C₅, or C₆).

The term “cycloalkyl” as used herein refers to a 3- to 14-memberedsaturated or unsaturated non-aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system. Included in this class are cycloalkyl groupswhich are fused to a benzene ring. Representative cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl,cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,1,3-cyclohexadienyl, cycloheptyl, cycloheptenyl, 1,3-cycloheptadienyl,1,4-cycloheptadienyl, -1,3,5-cycloheptatrienyl, cyclooctyl,cyclooctenyl, 1,3-cyclooctadienyl, 1,4-cyclooctadienyl,-1,3,5-cyclooctatrienyl, decahydronaphthalene, octahydronaphthalene,hexahydronaphthalene, octahydroindene, hexahydroindene, tetrahydroinden,decahydrobenzocycloheptene, octahydrobenzocycloheptene,hexahydrobenzocycloheptene, tetrahydrobenzocyclopheptene,dodecahydroheptalene, decahydroheptalene, octahydroheptalene,hexahydroheptalene, tetrahydroheptalene, (1s,3s)-bicyclo[1.1.0]butane,bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, Bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane,bicyclo[3.3.1]nonane, bicyclo[3.3.2]decane, bicyclo[3.3.]undecane,bicyclo[4.2.2]decane, and bicyclo[4.3.1]decane. A cycloalkyl group canbe unsubstituted or optionally substituted with one or more substituentsas described herein below.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.

The term “haloalkyl,” as used herein, refers to a C₁-C₆ alkyl groupwherein from one or more of the C₁-C₆ alkyl group's hydrogen atom isreplaced with a halogen atom, which can be the same or different.Examples of haloalkyl groups include, but are not limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl,pentachloroethyl, and 1,1,1-trifluoro-2-bromo-2-chloroethyl.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chainalkyl, or combinations thereof, consisting of carbon atoms and from oneto three heteroatoms selected from the group consisting of O, N, and S,and wherein the nitrogen and sulfur atoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. The heteroatom(s)O, N, and S can be placed at any position of the heteroalkyl group.Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, and—CH₂—CH═N—OCH₃. Up to two heteroatoms can be consecutive, for example,—CH₂—NH—OCH₃. When a prefix such as (C₂-C₈) is used to refer to aheteroalkyl group, the number of carbons (2 to 8, in this example) ismeant to include the heteroatoms as well. For example, a C₂-heteroalkylgroup is meant to include, for example, —CH₂OH (one carbon atom and oneheteroatom replacing a carbon atom) and —CH₂SH.

To further illustrate the definition of a heteroalkyl group, where theheteroatom is oxygen, a heteroalkyl group can be an oxyalkyl group. Forinstance, (C₂-C₅) oxyalkyl is meant to include, for example —CH₂—O—CH₃(a C₃-oxyalkyl group with two carbon atoms and one oxygen replacing acarbon atom), —CH₂CH₂CH₂CH₂OH, —OCH₂CH₂OCH₂CH₂OH,—OCH₂CH(OH)CH₂OH, andthe like.

The term “heteroaryl” as used herein refers to an aromatic heterocyclering of 5 to 14 members and having at least one heteroatom selected fromnitrogen, oxygen, and sulfur, and containing at least 1 carbon atom,including monocyclic, bicyclic, and tricyclic ring systems.Representative heteroaryls are triazolyl, tetrazolyl, oxadiazolyl,pyridyl, furyl, benzofuranyl, thienyl, benzothienyl, quinolinyl,pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl,phthalazinyl, quinazolinyl, pyrimidyl, azepinyl, oxepinyl, quinoxalinyland oxazolyl. A heteroaryl group can be unsubstituted or optionallysubstituted with one or more substituents as described herein below.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), and sulfur (S).

As used herein, the term “heterocycle” refers to 3- to 14-membered ringsystems which are either saturated, unsaturated, or aromatic, and whichcontains from 1 to 4 heteroatoms independently selected from nitrogen,oxygen, and sulfur, and wherein the nitrogen and sulfur heteroatoms canbe optionally oxidized, and the nitrogen heteroatom can be optionallyquaternized, including monocyclic, bicyclic, and tricyclic ring systems.The bicyclic and tricyclic ring systems may encompass a heterocycle orheteroaryl fused to a benzene ring. The heterocycle can be attached viaany heteroatom or carbon atom, where chemically acceptable. Heterocyclesinclude heteroaryls as defined above. Representative examples ofheterocycles include, but are not limited to, aziridinyl, oxiranyl,thiiranyl, triazolyl, tetrazolyl, azirinyl, diaziridinyl, diazirinyl,oxaziridinyl, azetidinyl, azetidinonyl, oxetanyl, thietanyl,piperidinyl, piperazinyl, morpholinyl, pyrrolyl, oxazinyl, thiazinyl,diazinyl, dioxanyl, triazinyl, tetrazinyl, imidazolyl, tetrazolyl,pyrrolidinyl, isoxazolyl, furanyl, furazanyl, pyridinyl, oxazolyl,benzoxazolyl, benzisoxazolyl, thiazolyl, benzthiazolyl, thienyl,pyrazolyl, triazolyl, pyrimidinyl, benzimidazolyl, isoindolyl,indazolyl, benzodiazolyl, benzotriazolyl, benzoxazolyl, benzisoxazolyl,purinyl, indolyl, isoquinolinyl, quinolinyl, and quinazolinyl. Aheterocycle group can be unsubstituted or optionally substituted withone or more substituents as described herein below.

The term “heterocycloalkyl,” by itself or in combination with otherterms, represents, unless otherwise stated, cyclic versions of“heteroalkyl.” Additionally, a heteroatom can occupy the position atwhich the heterocycle is attached to the remainder of the molecule.Examples of heterocycloalkyl include 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like.

The term “hydroxyalkyl,” as used herein, refers to an alkyl group havingthe indicated number of carbon atoms wherein one or more of the hydrogenatoms in the alkyl group is replaced with an —OH group. Examples ofhydroxyalkyl groups include, but are not limited to, —CH₂OH, —CH₂CH₂OH,—CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂CH₂OH, and branched versions thereof.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

As used herein, N-oxide, or amine oxide, refers to a compound derivedfrom a tertiary amine by the attachment of one oxygen atom to thenitrogen atom, R₃N⁺—O⁻. By extension the term includes the analogousderivatives of primary and secondary amines.

As used herein and unless otherwise indicated, the term “stereoisomer”means one stereoisomer of a compound that is substantially free of otherstereoisomers of that compound. For example, a stereomerically purecompound having one chiral center will be substantially free of theopposite enantiomer of the compound. A stereomerically pure compoundhaving two chiral centers will be substantially free of otherdiastereomers of the compound. In some embodiments, a stereomericallypure compound comprises greater than about 80% by weight of onestereoisomer of the compound and less than about 20% by weight of otherstereoisomers of the compound, for example greater than about 90% byweight of one stereoisomer of the compound and less than about 10% byweight of the other stereoisomers of the compound, or greater than about95% by weight of one stereoisomer of the compound and less than about 5%by weight of the other stereoisomers of the compound, or greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound.

As used herein, “protein” is used synonymously with “peptide,”“polypeptide,” or “peptide fragment”. A “purified” polypeptide, protein,peptide, or peptide fragment is substantially free of cellular materialor other contaminating proteins from the cell, tissue, or cell-freesource from which the amino acid sequence is obtained, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized.

As used herein, “modulate” is meant to refer to an increase or decreasein the levels of a peptide or a polypeptide, or to increase or decreasethe stability or activity of a peptide or a polypeptide. The term“inhibit” is meant to refer to a decrease in the levels of a peptide ora polypeptide or to a decrease in the stability or activity of a peptideor a polypeptide. In preferred embodiments, the peptide which ismodulated or inhibited is S-nitrosoglutathione (GSNO) or proteinS-nitrosothiols (SNOs).

As used here, the terms “nitric oxide” and “NO” encompass unchargednitric oxide and charged nitric oxide species, particularly includingnitrosonium ion (NO⁺) and nitroxyl ion (NO⁻). The reactive form ofnitric oxide can be provided by gaseous nitric oxide. Compounds havingthe structure X—NO_(y) wherein X is a nitric oxide releasing,delivering, or transferring moiety, including any and all such compoundswhich provide nitric oxide to its intended site of action in a formactive for their intended purpose, and Y is 1 or 2.

Repair” means recovering of structural integrity and normal physiologicfunction. By way of example, the oral and upper airway respiratoryepithelium can repair damage done by thermal injury or viral infection.

“Regeneration” means the ability of an organ to enter non-malignantcellular, vascular and stromal growth to restore functional organtissue. By way of example, wound healing involves regeneration of tissueand organs (e.g. skin, gastric and intestinal mucosa), as does bonefollowing fracture, and the liver following partial surgical removal andexposure to infectious or toxic insult.

As utilized herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of a federal or a state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals and, more particularly, in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered and includes, but is not limitedto such sterile liquids as water and oils.

A “pharmaceutically acceptable salt” or “salt” of a compound of theinvention is a product of the disclosed compound that contains an ionicbond, and is typically produced by reacting the disclosed compound witheither an acid or a base, suitable for administering to a subject. Apharmaceutically acceptable salt can include, but is not limited to,acid addition salts including hydrochlorides, hydrobromides, phosphates,sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates,arylalkylsulfonates, acetates, benzoates, citrates, maleates, fumarates,succinates, lactates, and tartrates; alkali metal cations such as Li,Na, and K, alkali earth metal salts such as Mg or Ca, or organic aminesalts.

A “pharmaceutical composition” is a formulation comprising the disclosedcompounds in a form suitable for administration to a subject. Apharmaceutical composition of the invention is preferably formulated tobe compatible with its intended route of administration. Examples ofroutes of administration include, but are not limited to, oral andparenteral, e.g., intravenous, intradermal, subcutaneous, inhalation,topical, transdermal, transmucosal, and rectal administration.

The term “substituted,” as used herein, means that any one or morehydrogens on the designated atom is replaced with a selection from theindicated group, provided that the designated atom's normal valency isnot exceeded, and that the substitution results in a stable compound.When a substituent is keto (i.e., ═O), then 2 hydrogens on the atom arereplaced. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N, or N═N).

Substituents for the groups referred to as alkyl, heteroalkyl, alkylene,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl can be selected from a variety of groups including—OR^(d), ═O, ═NR^(d)′, ═N—OR^(d), —NR^(d)′R^(d)″, —SR^(d)′, -halo,—SiR^(d)′R^(d)″R^(d)′″, —OC(O)R^(d)′, —C(O)R^(d)′, —CO₂R^(d)′,—CONR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″, —NR^(d)″C(O)R^(d)′,—NR^(d)′″C(O)NR^(d)′R^(d)″, —NR^(d)′″SO₂NR^(d)′R^(d)″,—NR^(d)″CO₂R^(d)′, —NHC(NH₂)═NH, —NR^(a)′C(NH₂)═NH, —NHC(NH₂)═NR^(d)′,—S(O)R^(d)′, —SO₂R^(d)′, —SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN, and—NO₂, in a number ranging from zero to three, with those groups havingzero, one or two substituents being exemplary.

R^(d)′, R^(d)″, and R^(d)′″ each independently refer to hydrogen,unsubstituted (C₁-C₈)alkyl, unsubstituted hetero(C₁-C₈) alkyl,unsubstituted aryl, and aryl substituted with one to three substituentsselected from -halo, unsubstituted alkyl, unsubstituted alkoxy,unsubstituted thioalkoxy, and unsubstituted aryl (C₁-C₄)alkyl. WhenR^(d)′ and R^(d)″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR^(d)′R^(d)″ can represent 1-pyrrolidinyl or4-morpholinyl.

Typically, an alkyl or heteroalkyl group will have from zero to threesubstituents, with those groups having two or fewer substituents beingexemplary of the present invention. An alkyl or heteroalkyl radical canbe unsubstituted or monosubstituted. In some embodiments, an alkyl orheteroalkyl radical will be unsubstituted.

Exemplary substituents for the alkyl and heteroalkyl radicals include,but are not limited to —OR^(d)′, ═O, ═NR^(d)′, ═N—OR^(d)′,—NR^(d)′R^(d)″, —SR^(d)′, -halo, —SiR^(d)′R^(d)″R^(d)′″, —OC(O)R^(d)′,—C(O)R^(d)′, —CO₂R^(d)′, —CONR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″,—NR^(d)″C(O)R^(d)′, —NR^(d)′″C(O)NR^(d)′R^(d)″,—NR^(d)′″SO₂NR^(d)′R^(d)″, —NR^(d)″CO₂R^(d)′, —NHC(NH₂)═NH,—NR^(a)′C(NH₂)═NH, —NHC(NH₂)═NR^(d)′, —S(O)R^(d)′, —SO₂R^(d)′,—SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN, and —NO₂, where R^(d)′,R^(d)″, and R^(d)′″ are as defined above. Typical substituents can beselected from: —OR″, ═O, —NR^(d)′R^(d)″, -halo, —OC(O)R^(d)′,—CO₂R^(d)′, —C(O)NR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″, —NR^(d)″C(O)R^(d)′,—NR^(d)″CO₂R^(d)′, —NR^(d)′″SO₂NR^(d)′R^(d)″-SO₂R^(d)′,—SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN, and —NO₂.

Similarly, substituents for the aryl and heteroaryl groups are variedand selected from: -halo, —OR^(e)′, —OC(O)R^(e)′, —NR^(e)′R^(e)″,—SR^(e)′, —R^(e)′, —CN, —NO₂, —CO₂R^(e)′, —C(O)NR^(e)′R^(e)″,—C(O)R^(e)′, —OC(O)NR^(e)′R^(e)″, —NR^(e)″C(O)R^(e)′, —NR^(e)″CO₂R^(e)′,—NR^(e)′″C(O)NR^(e)′R^(e)″, —NR^(e)′″SO₂NR^(e)′R^(e)″, —NHC(NH₂)═NH,—NR^(e)′C(NH₂)═NH, —NH—C(NH₂)═NR^(e)′, —S(O)R^(e)′, —SO₂R^(e)′,—SO₂NR^(e)′R^(e)″, —NR^(e)″SO₂R^(e)′, —N₃, —CH(Ph)₂, perfluoroalkoxy,and perfluoro(C₁-C₄)alkyl, in a number ranging from zero to the totalnumber of open valences on the aromatic ring system.

R^(e)′, R^(e)″ and R^(e)″′ are independently selected from hydrogen,unsubstituted (C₁-C₈) alkyl, unsubstituted hetero(C₁-C₈) alkyl,unsubstituted aryl, unsubstituted heteroaryl, unsubstituted aryl(C₁-C₄)alkyl, and unsubstituted aryloxy(C₁-C₄) alkyl. Typically, an aryl orheteroaryl group will have from zero to three substituents, with thosegroups having two or fewer substituents being exemplary in the presentinvention. In one embodiment of the invention, an aryl or heteroarylgroup will be unsubstituted or monosubstituted. In another embodiment,an aryl or heteroaryl group will be unsubstituted.

Two of the substituents on adjacent atoms of an aryl or heteroaryl ringin an aryl or heteroaryl group as described herein may optionally bereplaced with a substituent of the formula -T-C(O)—(CH₂)_(q)—U—, whereinT and U are independently —NH—, —O—, —CH₂— or a single bond, and q is aninteger of from 0 to 2. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -J-(CH₂)_(r)—K—, wherein J and K areindependently —CH₂—, —O—, —NH—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR^(f)′—, ora single bond, and r is an integer of from 1 to 3. One of the singlebonds of the new ring so formed may optionally be replaced with a doublebond. Alternatively, two of the substituents on adjacent atoms of thearyl or heteroaryl ring may optionally be replaced with a substituent ofthe formula —(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independentlyintegers of from 0 to 3, and X is —O—, —NR^(f)′—, —S—, —S(O)—, —S(O)₂—,or —S(O)₂NR^(a)′—. The substituent R^(f)′ in —NR^(f)′- and—S(O)₂NR^(f)′— is selected from hydrogen or unsubstituted (C₁-C₆) alkyl.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

As used herein the term “therapeutically effective amount” generallymeans the amount necessary to ameliorate at least one symptom of adisorder to be prevented, reduced, or treated as described herein. Thephrase “therapeutically effective amount” as it relates to the GSNORinhibitors of the present invention shall mean the GSNOR inhibitordosage that provides the specific pharmacological response for which theGSNOR inhibitor is administered in a significant number of subjects inneed of such treatment. It is emphasized that a therapeuticallyeffective amount of a GSNOR inhibitor that is administered to aparticular subject in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

The term “biological sample” includes, but is not limited to, samples ofblood (e.g., serum, plasma, or whole blood), urine, saliva, sweat,breast milk, vaginal secretions, semen, hair follicles, skin, teeth,bones, nails, or other secretions, body fluids, tissues, or cells. Inaccordance with the invention, the levels of the GSNOR in the biologicalsample can be determined by the methods described in U.S. PatentApplication Publication No. 2005/0014697.

D. Pharmaceutical Compositions

The invention encompasses pharmaceutical compositions comprising atleast one compound of the invention described herein and at least onepharmaceutically acceptable carrier. Suitable carriers are described in“Remington: The Science and Practice, Twentieth Edition,” published byLippincott Williams & Wilkins, which is incorporated herein byreference. Pharmaceutical compositions according to the invention mayalso comprise one or more non-inventive compound active agents.

The pharmaceutical compositions of the invention can comprise novelcompounds described herein, the pharmaceutical compositions can compriseknown compounds which previously were not known to have GSNOR inhibitoractivity, or a combination thereof.

The compounds of the invention can be utilized in any pharmaceuticallyacceptable dosage form, including, but not limited to injectable dosageforms, liquid dispersions, gels, aerosols, ointments, creams,lyophilized formulations, dry powders, tablets, capsules, controlledrelease formulations, fast melt formulations, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, mixed immediate release and controlled releaseformulations, etc. Specifically, the compounds of the inventiondescribed herein can be formulated: (a) for administration selected fromthe group consisting of oral, pulmonary, intravenous, intra-arterial,intrathecal, intra-articular, rectal, ophthalmic, colonic, parenteral,otic, intracisternal, intravaginal, intraperitoneal, local, buccal,nasal, and topical administration; (b) into a dosage form selected fromthe group consisting of liquid dispersions, gels, aerosols, ointments,creams, tablets, sachets, and capsules; (c) into a dosage form selectedfrom the group consisting of lyophilized formulations, dry powders, fastmelt formulations, controlled release formulations, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, and mixed immediate release and controlled releaseformulations; or (d) any combination thereof.

For respiratory infections, an inhalation formulation can be used toachieve high local concentrations. Formulations suitable for inhalationinclude dry power or aerosolized or vaporized solutions, dispersions, orsuspensions capable of being dispensed by an inhaler or nebulizer intothe endobronchial or nasal cavity of infected patients to treat upperand lower respiratory bacterial infections.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can comprise one or more of the followingcomponents: (1) a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycol,or other synthetic solvents; (2) antibacterial agents such as benzylalcohol or methyl parabens; (3) antioxidants such as ascorbic acid orsodium bisulfite; (4) chelating agents such asethylenediaminetetraacetic acid; (5) buffers such as acetates, citrates,or phosphates; and (5) agents for the adjustment of tonicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. A parenteral preparationcan be enclosed in ampoules, disposable syringes, or multiple dose vialsmade of glass or plastic.

Pharmaceutical compositions suitable for injectable use may comprisesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. The pharmaceutical composition should bestable under the conditions of manufacture and storage and should bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The carrier can be a solvent or dispersion medium comprising, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion, and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol or sorbitol, and inorganic saltssuch as sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activereagent in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating at least one compound of the invention into a sterilevehicle that contains a basic dispersion medium and any other requiredingredients. In the case of sterile powders for the preparation ofsterile injectable solutions, exemplary methods of preparation includevacuum drying and freeze-drying, both of which yield a powder of acompound of the invention plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed, for example, in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the compound of the invention can be incorporated withexcipients and used in the form of tablets, troches, or capsules. Oralcompositions can also be prepared using a fluid carrier for use as amouthwash, wherein the compound in the fluid carrier is applied orallyand swished and expectorated or swallowed. Pharmaceutically compatiblebinding agents, and/or adjuvant materials can be included as part of thecomposition.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, anebulized liquid, or a dry powder from a suitable device. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active reagents are formulated into ointments, salves, gels, orcreams as generally known in the art. The reagents can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In one embodiment, the compounds of the invention are prepared withcarriers that will protect against rapid elimination from the body. Forexample, a controlled release formulation can be used, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Additionally, suspensions of the compounds of the invention may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also include suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of thecompound of the invention calculated to produce the desired therapeuticeffect in association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the invention are dictated byand directly dependent on the unique characteristics of the compound ofthe invention and the particular therapeutic effect to be achieved, andthe limitations inherent in the art of compounding such an active agentfor the treatment of individuals.

Pharmaceutical compositions according to the invention comprising atleast one compound of the invention can comprise one or morepharmaceutical excipients. Examples of such excipients include, but arenot limited to binding agents, filling agents, lubricating agents,suspending agents, sweeteners, flavoring agents, preservatives, buffers,wetting agents, disintegrants, effervescent agents, and otherexcipients. Such excipients are known in the art. Exemplary excipientsinclude: (1) binding agents which include various celluloses andcross-linked polyvinylpyrrolidone, microcrystalline cellulose, such asAvicel® PH101 and Avicel® PH102, silicified microcrystalline cellulose(ProSolv SMCC™), gum tragacanth and gelatin; (2) filling agents such asvarious starches, lactose, lactose monohydrate, and lactose anhydrous;(3) disintegrating agents such as alginic acid, Primogel, corn starch,lightly crosslinked polyvinyl pyrrolidone, potato starch, maize starch,and modified starches, croscarmellose sodium, cross-povidone, sodiumstarch glycolate, and mixtures thereof; (4) lubricants, including agentsthat act on the flowability of a powder to be compressed, includemagnesium stearate, colloidal silicon dioxide, such as Aerosil® 200,talc, stearic acid, calcium stearate, and silica gel; (5) glidants suchas colloidal silicon dioxide; (6) preservatives, such as potassiumsorbate, methylparaben, propylparaben, benzoic acid and its salts, otheresters of parahydroxybenzoic acid such as butylparaben, alcohols such asethyl or benzyl alcohol, phenolic compounds such as phenol, orquaternary compounds such as benzalkonium chloride; (7) diluents such aspharmaceutically acceptable inert fillers, such as microcrystallinecellulose, lactose, dibasic calcium phosphate, saccharides, and/ormixtures of any of the foregoing; examples of diluents includemicrocrystalline cellulose, such as Avicel® PH101 and Avicel® PH102;lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose®DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch;sorbitol; sucrose; and glucose; (8) sweetening agents, including anynatural or artificial sweetener, such as sucrose, saccharin sucrose,xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9)flavoring agents, such as peppermint, methyl salicylate, orangeflavoring, Magnasweet® (trademark of MAFCO), bubble gum flavor, fruitflavors, and the like; and (10) effervescent agents, includingeffervescent couples such as an organic acid and a carbonate orbicarbonate. Suitable organic acids include, for example, citric,tartaric, malic, fumaric, adipic, succinic, and alginic acids andanhydrides and acid salts. Suitable carbonates and bicarbonates include,for example, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate, magnesium carbonate, sodium glycine carbonate,L-lysine carbonate, and arginine carbonate. Alternatively, only thesodium bicarbonate component of the effervescent couple may be present.

E. Kits Comprising the Compositions of the Invention

The present invention also encompasses kits comprising the compositionsof the invention. Such kits can comprise, for example, (1) at least onecompound of the invention; and (2) at least one pharmaceuticallyacceptable carrier, such as a solvent or solution. Additional kitcomponents can optionally include, for example: (1) any of thepharmaceutically acceptable excipients identified herein, such asstabilizers, buffers, etc., (2) at least one container, vial, or similarapparatus for holding and/or mixing the kit components; and (3) deliveryapparatus, such as an inhaler, nebulizer, syringe, etc.

F. Methods of Preparing Compounds of the Invention

The compounds of the invention can readily be synthesized using knownsynthetic methodologies or via a modification of known syntheticmethodologies. As would be readily recognized by a skilled artisan, themethodologies described below allow the synthesis of bicyclic aromaticcompounds having a variety of substituents. Exemplary synthetic methodsare described in the Examples section below.

If needed, further purification and separation of enantiomers anddiastereomers can be achieved by routine procedures known in the art.Thus, for example, the separation of enantiomers of a compound can beachieved by the use of chiral HPLC and related chromatographictechniques. Diastereomers can be similarly separated. In some instances,however, diastereomers can simply be separated physically, such as, forexample, by controlled precipitation or crystallization.

The process of the invention, when carried out as prescribed herein, canbe conveniently performed at temperatures that are routinely accessiblein the art. In one embodiment, the process is performed at a temperaturein the range of about 25° C. to about 110° C. In another embodiment, thetemperature is in the range of about 40° C. to about 100° C. In yetanother embodiment, the temperature is in the range of about 50° C. toabout 95° C.

Synthetic steps that require a base are carried out using any convenientorganic or inorganic base. Typically, the base is not nucleophilic.Thus, in one embodiment, the base is selected from carbonates,phosphates, hydroxides, alkoxides, salts of disilazanes, and tertiaryamines.

The process of the invention, when performed as described herein, can besubstantially complete after several minutes to after several hoursdepending upon the nature and quantity of reactants and reactiontemperature. The determination of when the reaction is substantiallycomplete can be conveniently evaluated by ordinary techniques known inthe art such as, for example, HPLC, LCMS, TLC, and ¹H NMR.

G. Methods of Treatment

The invention encompasses methods of preventing or treating (e.g.,alleviating one or more symptoms of) medical conditions through use ofone or more of the disclosed compounds. The methods compriseadministering a therapeutically effective amount of a compound of theinvention to a patient in need. The compositions of the invention canalso be used for prophylactic therapy.

The compound of the invention used in the methods of treatment accordingto the invention can be: (1) a novel compound described herein, or apharmaceutically acceptable salt thereof, a stereoisomer thereof, aprodrug thereof, a metabolite thereof, or an N-oxide thereof; (2) acompound which was known prior to the present invention, but wherein itwas not known that the compound is a GSNOR inhibitor, or apharmaceutically acceptable salt thereof, a stereoisomer thereof, aprodrug thereof, a metabolite thereof, or an N-oxide thereof; or (3) acompound which was known prior to the present invention, and wherein itwas known that the compound is a GSNOR inhibitor, but wherein it was notknown that the compound is useful for the methods of treatment describedherein, or a pharmaceutically acceptable salt, a stereoisomer, aprodrug, a metabolite, or an N-oxide thereof.

The patient can be any animal, domestic, livestock, or wild, including,but not limited to cats, dogs, horses, pigs, and cattle, and preferablyhuman patients. As used herein, the terms patient and subject may beused interchangeably.

As used herein, “treating” describes the management and care of apatient for the purpose of combating a disease, condition, or disorderand includes the administration of a compound of the present inventionto prevent the onset of the symptoms or complications, alleviating thesymptoms or complications, or eliminating the disease, condition, ordisorder. More specifically, “treating” includes reversing, attenuating,alleviating, minimizing, suppressing, or halting at least onedeleterious symptom or effect of a disease (disorder) state, diseaseprogression, disease causative agent (e.g., bacteria or viruses), orother abnormal condition. Treatment is continued as long as symptomsand/or pathology ameliorate.

In general, the dosage, i.e., the therapeutically effective amount,ranges from 1 μg/kg to 10 g/kg and often ranges from 10 μg/kg to 1 g/kgor 10 μg/kg to 100 mg/kg body weight of the subject being treated, perday.

H. GSNOR Uses

In subjects with deleteriously high levels of GSNOR or GSNOR activity,modulation may be achieved, for example, by administering one or more ofthe disclosed compounds that disrupts or down-regulates GSNOR function,or decreases GSNOR levels. These compounds may be administered withother GSNOR inhibitor agents, such as anti-GSNOR antibodies or antibodyfragments, GSNOR antisense, iRNA, or small molecules, or otherinhibitors, alone or in combination with other agents as described indetail herein.

The present invention provides a method of treating a subject afflictedwith a disorder ameliorated by NO donor therapy. Such a method comprisesadministering to a subject a therapeutically effective amount of a GSNORinhibitor.

The disorders can include pulmonary disorders associated with hypoxemiaand/or smooth muscle constriction in the lungs and airways and/or lunginfection and/or lung inflammation and/or lung injury (e.g., pulmonaryhypertension, ARDS, asthma, pneumonia, pulmonary fibrosis/interstitiallung diseases, cystic fibrosis, COPD); cardiovascular disease and heartdisease (e.g., hypertension, ischemic coronary syndromes,atherosclerosis, heart failure, glaucoma); diseases characterized byangiogenesis (e.g., coronary artery disease); disorders where there isrisk of thrombosis occurring; disorders where there is risk ofrestenosis occurring; inflammatory diseases (e.g., AIDS relateddementia, inflammatory bowel disease (IBD), Crohn's disease, colitis,and psoriasis); functional bowel disorders (e.g., irritable bowelsyndrome (IBS)); diseases where there is risk of apoptosis occurring(e.g., heart failure, atherosclerosis, degenerative neurologicdisorders, arthritis, and liver injury (e.g., drug induced, ischemic oralcoholic)); impotence; sleep apnea; diabetic wound healing; cutaneousinfections; treatment of psoriasis; obesity caused by eating in responseto craving for food; stroke; reperfusion injury (e.g., traumatic muscleinjury in heart or lung or crush injury); and disorders wherepreconditioning of heart or brain for NO protection against subsequentischemic events is beneficial, central nervous system (CNS) disorders(e.g., anxiety, depression, psychosis, and schizophrenia); andinfections caused by bacteria (e.g., tuberculosis, C. difficileinfections, among others).

In one embodiment, the disorder is liver injury. Liver injury caninclude, for example, acute liver toxicity. Acute liver toxicity canresult in acute liver failure. Acute liver failure (ALF) is an uncommonbut potentially lethal drug-related adverse effect that often leads toliver transplantation (LT) or death. Acetoaminophen is the most commoncause of acute liver toxicity and acute liver failure, although acuteliver toxicity can be due to other agents, such as alcohol and otherdrugs. Regardless of whether it occurs as a result of a single overdoseor after repeated supratherapeutic ingestion, the progression ofacetaminophen poisoning can be categorized into four stages: preclinicaltoxic effects (a normal serum alanine aminotransferase concentration),hepatic injury (an elevated alanine aminotransferase concentration),hepatic failure (hepatic injury with hepatic encephalopathy), andrecovery. As long as sufficient glutathione is present, the liver isprotected from injury. Overdoses of acetaminophen (either a single largeingestion or repeated supratherapeutic ingestion) can deplete hepaticglutathione stores and allow liver injury to occur. Compounds of theinvention are capable of treating and/or preventing liver injury and/oracute liver toxicity. In this embodiment, appropriate amounts ofcompounds of the present invention are an amount sufficient to treatand/or prevent liver injury and can be determined without undueexperimentation by preclinical and/or clinical trials. In oneembodiment, the amount to treat is at least 0.001 mg/kg, at least 0.002mg/kg, at least 0.003 mg/kg, at least 0.004 mg/kg, at least 0.005 mg/kg,at least 0.006 mg/kg, at least 0.007 mg/kg, at least 0.008 mg/kg, atleast 0.009 mg/kg, at least 0.01 mg/kg, at least 0.02 mg/kg, at least0.03 mg/kg, at least 0.04 mg/kg, at least 0.05 mg/kg, at least at least0.06 mg/kg, at least 0.07 mg/kg, at least 0.08 mg/kg, at least 0.09mg/kg, at least 0.1 mg/kg, at least 0.2 mg/kg, at least 0.3 mg/kg, atleast 0.4 mg/kg, at least 0.5 mg/kg, at least 0.6 mg/kg, at least 0.7mg/kg, at least 0.8 mg/kg, at least 0.9 mg/kg, at least 1 mg/kg, atleast 1.5 mg/kg, at least 2 mg/kg, at least 2.5 mg/kg, at least 3 mg/kg,at least 3.5 mg/kg, at least 4 mg/kg, at least 4.5 mg/kg, at least 5mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least30 mg/kg, at least 40 mg/kg, at least 50 mg/kg, at least 60 mg/kg, atleast 70 mg/kg, at least 80 mg/kg, at least 90 mg/kg, at least 100mg/kg. The dosing can be hourly, four times, twice, or once daily, orfour times, twice, or once per week, or weekly, or every other week,every third week, or monthly.

In one embodiment, the disorder is trauma (including surgery andthermal), infectious, toxic, aging, and ischemic damage to organs ofknown regenerative capacity, such as skin, gastric mucosa, airwayepithelial and cartilaginous structures, liver, neuronal structures suchas the spinal cord, bone marrow and bone. We have shown that inhibitionof GSNOR by the use of highly specific small molecules treats, repairs,and promotes regeneration of mammalian tissue. By way of example, smallmolecule inhibitors are effective in treating, and promoting repair andregeneration of mammalian lung tissue damaged by instillation of achemical agent known to cause severe lung injury (porcine pancreaticelastase) (Blonder et al., ATS 2011 abstract reference). In thisembodiment, appropriate amounts of compounds of the present inventionare an amount sufficient to regenerate tissue/organs and can bedetermined without undue experimentation by preclinical and/or clinicaltrials.

In one embodiment the disorder is trauma (including surgery andthermal), infectious, toxic, aging, and ischemic damage to organs of notcommonly known to have regenerative capacity. Examples includeregeneration of: the heart, the lung, the kidney, the central nervoussystem, the peripheral nervous system, peripheral vascular tissue,liver, pancreas, adrenal gland, thyroid, testes, ovary, retina, tongue,bone, bladder, esophagus, larynx, thymus, spleen, cartilaginousstructures of the head, and cartilaginous structures of the joints. Inthis embodiment, appropriate amounts of compounds of the presentinvention are an amount sufficient to regenerate tissue/organs and canbe determined without undue experimentation by preclinical and/orclinical trials.

In one embodiment ex and in vivo implantation and regeneration of organsand structures, including stem cells. In this embodiment, appropriateamounts of compounds of the present invention are an amount sufficientto regenerate tissue/organs and can be determined without undueexperimentation by preclinical and/or clinical trials.

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, or a prodrug, stereoisomer,metabolite, or N-oxide thereof, can be administered in combination withan NO donor. An NO donor donates nitric oxide or a related redox speciesand more generally provides nitric oxide bioactivity, that is activitywhich is identified with nitric oxide, e.g., vasorelaxation orstimulation or inhibition of a receptor protein, e.g., ras protein,adrenergic receptor, NFκB. NO donors including S-nitroso, O-nitroso,C-nitroso, and N-nitroso compounds and nitro derivatives thereof andmetal NO complexes, but not excluding other NO bioactivity generatingcompounds, useful herein are described in “Methods in Nitric OxideResearch,” Feelisch et al. eds., pages 71-115 (J. S., John Wiley & Sons,New York, 1996), which is incorporated herein by reference. NO donorswhich are C-nitroso compounds where nitroso is attached to a tertiarycarbon which are useful herein include those described in U.S. Pat. No.6,359,182 and in WO 02/34705. Examples of S-nitroso compounds, includingS-nitrosothiols useful herein, include, for example,S-nitrosoglutathione, S-nitroso-N-acetylpenicillamine,S-nitroso-cysteine and ethyl ester thereof, S-nitroso cysteinyl glycine,S-nitroso-gamma-methyl-L-homocysteine, S-nitroso-L-homocysteine,S-nitroso-gamma-thio-L-leucine, S-nitroso-delta-thio-L-leucine, andS-nitrosoalbumin. Examples of other NO donors useful herein are sodiumnitroprusside (nipride), ethyl nitrite, isosorbide, nitroglycerin, SIN 1which is molsidomine, furoxamines, N-hydroxy (N-nitrosamine), andperfluorocarbons that have been saturated with NO or a hydrophobic NOdonor.

The combination of a GSNOR inhibitor with R(+) enantiomer of amlodipine,a known NO releaser (Zhang at al., J. Cardiovasc. Pharm. 39: 208-214(2002)) is also an embodiment of the present invention.

The present invention also provides a method of treating a subjectafflicted with pathologically proliferating cells where the methodcomprises administering to said subject a therapeutically effectiveamount of an inhibitor of GSNOR. The inhibitors of GSNOR are thecompounds as defined above, or a pharmaceutically acceptable saltthereof, or a stereoisomer, prodrug, metabolite, or N-oxide thereof, incombination with a pharmaceutically acceptable carrier. Treatment iscontinued as long as symptoms and/or pathology ameliorate.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating microbes. The microbes involved can bethose where GSNOR is expressed to protect the microbe from nitrosativestress or where a host cell infected with the microbe expresses theenzyme, thereby protecting the microbe from nitrosative stress. The term“pathologically proliferating microbes” is used herein to meanpathologic microorganisms including, but not limited to, pathologicbacteria, pathologic viruses, pathologic Chlamydia, pathologic protozoa,pathologic Rickettsia, pathologic fungi, and pathologic mycoplasmata.More detail on the applicable microbes is set forth at columns 11 and 12of U.S. Pat. No. 6,057,367. The term “host cells infected withpathologic microbes” includes not only mammalian cells infected withpathologic viruses but also mammalian cells containing intracellularbacteria or protozoa, e.g., macrophages containing Mycobacteriumtuberculosis, Mycobacterium leper (leprosy), or Salmonella typhi(typhoid fever).

In another embodiment, the pathologically proliferating cells can bepathologic helminths. The term “pathologic helminths” is used herein torefer to pathologic nematodes, pathologic trematodes and pathologiccestodes. More detail on the applicable helminths is set forth at column12 of U.S. Pat. No. 6,057,367.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating mammalian cells. The term “pathologicallyproliferating mammalian cells” as used herein means cells of the mammalthat grow in size or number in said mammal so as to cause a deleteriouseffect in the mammal or its organs. The term includes, for example, thepathologically proliferating or enlarging cells causing restenosis, thepathologically proliferating or enlarging cells causing benign prostatichypertrophy, the pathologically proliferating cells causing myocardialhypertrophy, and proliferating cells at inflammatory sites such assynovial cells in arthritis or cells associated with a cellproliferation disorder.

As used herein, the term “cell proliferative disorder” refers toconditions in which the unregulated and/or abnormal growth of cells canlead to the development of an unwanted condition or disease, which canbe cancerous or non-cancerous, for example a psoriatic condition. Asused herein, the term “psoriatic condition” refers to disordersinvolving keratinocyte hyperproliferation, inflammatory cellinfiltration, and cytokine alteration. The cell proliferative disordercan be a precancerous condition or cancer. The cancer can be primarycancer or metastatic cancer, or both.

As used herein, the term “cancer” includes solid tumors, such as lung,breast, colon, ovarian, pancreas, prostate, adenocarcinoma, squamouscarcinoma, sarcoma, malignant glioma, leiomyosarcoma, hepatoma, head andneck cancer, malignant melanoma, non-melanoma skin cancers, as well ashematologic tumors and/or malignancies, such as leukemia, childhoodleukemia and lymphomas, multiple myeloma, Hodgkin's disease, lymphomasof lymphocytic and cutaneous origin, acute and chronic leukemia such asacute lymphoblastic, acute myelocytic, or chronic myelocytic leukemia,plasma cell neoplasm, lymphoid neoplasm, and cancers associated withAIDS.

In addition to psoriatic conditions, the types of proliferative diseaseswhich may be treated using the compositions of the present invention areepidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneoushemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas,myofibromatosis, osteoplastic tumors, and other dysplastic masses, andthe like. In one embodiment, proliferative diseases include dysplasiasand disorders of the like.

In one embodiment, treating cancer comprises a reduction in tumor size,decrease in tumor number, a delay of tumor growth, decrease inmetastatic lesions in other tissues or organs distant from the primarytumor site, an improvement in the survival of patients, or animprovement in the quality of patient life, or at least two of theabove.

In another embodiment, treating a cell proliferative disorder comprisesa reduction in the rate of cellular proliferation, reduction in theproportion of proliferating cells, a decrease in size of an area or zoneof cellular proliferation, or a decrease in the number or proportion ofcells having an abnormal appearance or morphology, or at least two ofthe above.

In yet another embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a stereoisomer thereof, aprodrug thereof, a metabolite thereof, or an N-oxide thereof can beadministered in combination with a second chemotherapeutic agent. In afurther embodiment, the second chemotherapeutic agent is selected fromthe group consisting of tamoxifen, raloxifene, anastrozole, exemestane,letrozole, cisplatin, carboplatin, paclitaxel, cyclophosphamide,lovastatin, minosine, gemcitabine, araC, 5-fluorouracil, methotrexate,docetaxel, goserelin, vincristin, vinblastin, nocodazole, teniposide,etoposide, epothilone, navelbine, camptothecin, daunonibicin,dactinomycin, mitoxantrone, amsacrine, doxorubicin, epirubicin,idarubicin imatanib, gefitinib, erlotinib, sorafenib, sunitinib malate,trastuzumab, rituximab, cetuximab, and bevacizumab.

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a stereoisomer thereof, aprodrug thereof, a metabolite thereof, or an N-oxide thereof, can beadministered in combination with an agent that imposes nitrosative oroxidative stress. Agents for selectively imposing nitrosative stress toinhibit proliferation of pathologically proliferating cells incombination therapy with GSNOR inhibitors herein and dosages and routesof administration therefor include those disclosed in U.S. Pat. No.6,057,367, which is incorporated herein. Supplemental agents forimposing oxidative stress (i.e., agents that increase GSSG (oxidizedglutathione) over GSH (glutathione) ratio or NAD(P) over NAD(P)H ratioor increase thiobarbituric acid derivatives) in combination therapy withGSNOR inhibitors herein include, for example, L-buthionine-S-sulfoximine(BSO), glutathione reductase inhibitors (e.g., BCNU), inhibitors oruncouplers of mitochondrial respiration, and drugs that increasereactive oxygen species (ROS), e.g., adriamycin, in standard dosageswith standard routes of administration.

GSNOR inhibitors may also be co-administered with a phosphodiesteraseinhibitor (e.g., rolipram, cilomilast, roflumilast, Viagra® (sildenifilcitrate), Cialis® (tadalafil), Levitra® (vardenifil), etc.), aβ-agonist, a steroid, an anti-muscarinics, or a leukotriene antagonist(LTD-4). Those skilled in the art can readily determine the appropriatetherapeutically effective amount depending on the disorder to beameliorated.

GSNOR inhibitors may be used as a means to improve β-adrenergicsignaling. In particular, inhibitors of GSNOR alone or in combinationwith β-agonists could be used to treat or protect against heart failure,or other vascular disorders such as hypertension and asthma. GSNORinhibitors can also be used to modulate G protein coupled receptors(GPCRs) by potentiating Gs G-protein, leading to smooth musclerelaxation (e.g., airway and blood vessels), and by attenuating GqG-protein, and thereby preventing smooth muscle contraction (e.g., inairway and blood vessels).

The therapeutically effective amount for the treatment of a subjectafflicted with a disorder ameliorated by NO donor therapy is the GSNORinhibiting amount in vivo that causes amelioration of the disorder beingtreated or protects against a risk associated with the disorder. Forexample, for asthma, a therapeutically effective amount is abronchodilating effective amount; for cystic fibrosis, a therapeuticallyeffective amount is an airway obstruction ameliorating effective amount;for ARDS, a therapeutically effective amount is a hypoxemia amelioratingeffective amount; for heart disease, a therapeutically effective amountis an angina relieving or angiogenesis inducing effective amount; forhypertension, a therapeutically effective amount is a blood pressurereducing effective amount; for ischemic coronary disorders, atherapeutic amount is a blood flow increasing effective amount; foratherosclerosis, a therapeutically effective amount is an endothelialdysfunction reversing effective amount; for glaucoma, a therapeuticamount is an intraocular pressure reducing effective amount; fordiseases characterized by angiogenesis, a therapeutically effectiveamount is an angiogenesis inhibiting effective amount; for disorderswhere there is risk of thrombosis occurring, a therapeutically effectiveamount is a thrombosis preventing effective amount; for disorders wherethere is risk of restenosis occurring, a therapeutically effectiveamount is a restenosis inhibiting effective amount; for chronicinflammatory diseases, a therapeutically effective amount is aninflammation reducing effective amount; for disorders where there isrisk of apoptosis occurring, a therapeutically effective amount is anapoptosis preventing effective amount; for impotence, a therapeuticallyeffective amount is an erection attaining or sustaining effectiveamount; for obesity, a therapeutically effective amount is a satietycausing effective amount; for stroke, a therapeutically effective amountis a blood flow increasing or a TIA protecting effective amount; forreperfusion injury, a therapeutically effective amount is a functionincreasing effective amount; and for preconditioning of heart and brain,a therapeutically effective amount is a cell protective effectiveamount, e.g., as measured by troponin or CPK.

The therapeutically effective amount for the treatment of a subjectafflicted with pathologically proliferating cells means a GSNORinhibiting amount in vivo which is an antiproliferative effectiveamount. Such antiproliferative effective amount as used herein means anamount causing reduction in rate of proliferation of at least about 20%,at least about 10%, at least about 5%, or at least about 1%.

I. Uses in an Apparatus

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a stereoisomer, prodrug, metabolite, or N-oxidethereof, can be applied to various apparatus in circumstances when thepresence of such compounds would be beneficial. Such apparatus can beany device or container, for example, implantable devices in which acompound of the invention can be used to coat a surgical mesh orcardiovascular stent prior to implantation in a patient. The compoundsof the invention can also be applied to various apparatus for in vitroassay purposes or for culturing cells.

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a stereoisomer, a prodrug, a metabolite, or an N-oxidethereof, can also be used as an agent for the development, isolation orpurification of binding partners to compounds of the invention, such asantibodies, natural ligands, and the like. Those skilled in the art canreadily determine related uses for the compounds of the presentinvention.

EXAMPLES

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including a U.S. patent, are specificallyincorporated by reference.

Examples 1-71 list representative novel analogs of Formula I useful asGSNOR inhibitors of the invention. Exemplary schemes below illustratesome general methods of making analogs of Formula I. Synthetic methodsthat can be used to prepare each compound are described in Examples1-71. Supporting mass spectrometry data and/or proton NMR data for eachcompound is also included in Examples 1-71. Synthetic details forcorresponding Intermediates are detailed in Example 72.

Schemes 1-6 below illustrate general methods for preparing analogs.

For a detailed example of General Scheme 1 see Compound IV-1 in Example1.

For a detailed example of Scheme 2, A conditions, see Compound IV-2 inExample 2.

For a detailed example of Scheme 2, B conditions, see Compound IV-8 inExample 8.

For a detailed example of Scheme 3, see Compound IV-9 in Example 9.

For a detailed example of Scheme 4, Route A, see Compound IV-11 inExample 11.

For a detailed example of Scheme 4, Route B, see Compound IV-12 inExample 12.

For a detailed example of Scheme 5, Compound A, see Compound IV-33 inExample 33.

For a detailed example of Scheme 5, Compound B, see Compound IV-24 inExample 24.

For a detailed example of Scheme 5, Compound C, see Compound IV-23 inExample 23.

For a detailed example of Scheme 6, A conditions, see Compound V-1 inExample 45.

For a detailed example of Scheme 6, B conditions, see Compound V-2 inExample 46.

Example 1 Compound IV-1: 4-(6-hydroxy-3-methylquinolin-2-yl)benzoic acid

Followed Scheme 1

Step 1: Synthesis of Methyl 4-(6-methoxy-3-methylquinolin-2-yl)benzoate

To a mixture of 2-chloro-6-methoxy-3-methylquinoline (100 mg, 0.482mmol), 4-(methoxycarbonyl)phenylboronic acid (184 mg, 1.02 mmol), TEA(0.35 mL, 2.41 mmol), and PdCl₂(dppf) (35 mg, 0.048 mmol) was added 2 mLof DMF under argon. The mixture was then stirred for 2.5 h at 120° C. ina microwave reactor. The crude mixture was then diluted with water (25mL) and extracted with EtOAc (25 mL×2). The organics were washed withbrine (50 mL), dried over sodium sulfate, and concentrated to yield 250mg of crude material. The crude was purified via column chromatographywith a gradient of 5% EtOAc in hexanes to 80% EtOAc in hexanes to yield37 mg (25% yield) of methyl 4-(6-methoxy-3-methylquinolin-2-yl)benzoate.

Step 2: Synthesis of 4-(6-hydroxy-3-methylquinolin-2-yl)benzoic acid

Methyl 4-(6-methoxy-3-methylquinolin-2-yl)benzoate (37 mg, 0.121 mmol)was dissolved in 2 mL of DCM and BBr₃ (150 μL) was added. The mixturewas stirred at room temperature for 1 day followed by addition of 10 mLof H₂O. The solution was stirred vigorously for 1 h followed byfiltration of the solids. The solids were washed with H₂O and dried invacuo to yield 9.5 mg (28% yield) of Compound IV-1. ¹H NMR (DMSO-d₆, 400MHz): δ 8.37 (s, 1H), 8.10 (d, 2H), 7.94 (d, 1H), 7.78 (d, 2H),7.42-7.39 (dd, 1H), 7.24-7.23 (d, 1H), 2.43 (s, 3H). MS (ESI): m/z280.10 [M+H]⁺.

Example 2 Compound IV-2:2-(4-(1H-tetrazol-5-yl)phenyl)-3-methylquinolin-6-ol

Followed Scheme 2: A Conditions

Step 1: Synthesis of2-(4-(1H-tetrazol-5-yl)phenyl)-6-methoxy-3-methylquinoline

To a mixture of 2-chloro-6-methoxy-3-methylquinoline (100 mg, 0.482mmol), 4-(1H-tetrazol-5-yl)phenylboronic acid (91.2 mg, 0.482 mmol),K₂CO₃ (199 mg, 1.45 mmol), and PdCl₂(dppf) (17.6 mg, 0.024 mmol) wasadded 7 mL of DEGME and 3 mL of H₂O under argon. The mixture was stirredat 150° C. in a microwave reactor for 1.5 hours. The crude mixture wasdiluted with 1N NaOH (10 mL) and slowly acidified to a pH of 4.0 usingconc. HCl. The solids were filtered to yield 128 mg (84% yield) of thedesired product.

Step 2: Synthesis of(2-(4-(1H-tetrazol-5-yl)phenyl)-3-methylquinolin-6-ol)

2-(4-(1H-tetrazol-5-yl)phenyl)-6-methoxy-3-methylquinoline (128 mg, 0.40mmol) was dissolved in 5 mL of NMP and Na₂S (47 mg, 0.60 mmol) was addedto it. The mixture was then stirred for 4 hours at 140° C. in amicrowave reactor. After concentration in vacuo the crude was dissolvedin 5 mL of 1N NaOH and slowly acidified with 1N HCl to a pH of 4. Thesolids were filtered and dried in vacuo to afford 46.2 mg (38% yield) ofCompound IV-2. ¹H NMR (DMSO-d₆, 400 MHz): δ 10.10-10.00 (bs, 1H),8.18-8.15 (d, 2H), 8.08 (s, 1H), 7.87-7.84 (m, 3H), 7.30-7.26 (d, 1H),7.13 (s, 1H), 2.45 (s, 3H). MS (ESI): m/z 304.11 [M+H]⁺.

Example 3 Compound IV-3: 4-(6-hydroxyquinolin-2-yl)benzoic acid

Followed Scheme 2, A conditions: Starting materials:2-chloro-6-methoxyquinoline (Intermediate 1) (100 mg, 0.52 mmol) and(4-(methoxycarbonyl)phenyl) boronic acid. ¹H NMR (DMSO-d₆, 400 MHz): δ13.06-12.90 (bs, 1H), 10.14 (s, 1H), 8.36-8.33 (d, 2H), 8.30-8.27 (d,1H), 8.11-8.06 (m, 3H), 7.97-7.94 (d, 1H), 7.38-7.34 (dd, 1H), 7.20 (s,1H). MS (ESI): m/z 266.08 [M+H]⁺.

Example 4 Compound IV-4: 2-(4-(1H-tetrazol-5-yl)phenyl)quinolin-6-ol

Followed Scheme 2, A conditions: Starting materials:2-chloro-6-methoxyquinoline (Intermediate 1) and(4-(1H-tetrazol-5-yl)phenyl)boronic acid. ¹H NMR (DMSO-d₆, 400 MHz): δ10.15 (s, 1H), 8.45 (d, 2H), 8.31-8.28 (d, 1H), 8.22-8.12 (m, 3H),7.98-7.95 (d, 1H), 7.39-7.35 (dd, 1H), 7.21 (s, 1H). MS (ESI): m/z290.08 [M+H]⁺.

Example 5 Compound IV-5:1-(6-hydroxyquinolin-2-yl)piperidine-4-carboxylic acid

Step 1: Synthesis of ethyl 4-(6-methoxyquinolin-2-yl)cyclohexanecarboxylate

Ethyl piperidine-4-carboxylate (100 mg) was treated with2-chloro-6-methoxyquinoline (Intermediate 1) (90 mg) in MeCN (0.8 mL)and TEA (100 mg) in a sealed tube at 180° C. for 6 h in a microwavereactor. After aqueous work-up with EtOAc and column purification,eluting with EtOAc/hexane, ethyl4-(6-methoxyquinolin-2-yl)cyclohexanecarboxylate (90 mg) was afforded asa solid.

Step 2: Synthesis of Compound IV-5

Ethyl 4-(6-methoxyquinolin-2-yl)cyclohexanecarboxylate (90 mg) wastreated with sodium ethanethiolate (150 mg) in NMP (2 mL) at 100° C.over 48 h. The desired product (50 mg) was purified by a Dowex 50W X8cation exchange column, eluting with water and 2N NH₄OH solution. ¹H NMR(DMSO-d₆, 300 MHz): δ 7.82 (1H, d, J=9 Hz), 7.42 (1H, d, J=9 hz), 7.13(1H, d, J=9 Hz), 7.08 (1H, dd, J=3, 9 Hz), 6.93 (1H, d, J=3 Hz), 4.2-4.4(2H, m), 2.88-2.97 (2H, m), 2.15-2.21 (1H, m), 1.80-1.86 (2H, m),1.45-1.60 (1H, m) ppm. MS (ESI): m/z 273.0 [M+1]⁺.

Example 6 Compound IV-6:(1r,4r)-4-(6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid

Step 1: Synthesis of (1r,4r)-methyl4-(6-methoxyquinolin-2-yl)cyclohexanecarboxylate and (1s,4s)-methyl4-(6-methoxyquinolin-2-yl)cyclohexanecarboxylate

Methyl 4-(6-hydroxyquinolin-2-yl)cyclohex-3-enecarboxylate (261 mg)(Compound IV-16) was dissolved in EtOAc (10 mL) and mixed with 10% Pd/C(38.5 mg). The system was vacuumed shortly and charged with hydrogen.This procedure was repeated three times. The reaction mixture wasstirred under hydrogen for 3 h. After filtration to remove the catalystand concentration under reduced pressure, the resultant mixtures wereseparated by flash silica gel chromatography eluting with EtOAc/hexaneto afford (1s,4s)-methyl4-(6-methoxyquinolin-2-yl)cyclohexanecarboxylate (153 mg) and(1r,4r)-methyl 4-(6-methoxyquinolin-2-yl)cyclohexanecarboxylate (72 mg)separately.

Step 2: Synthesis of Compound IV-6

Followed the procedure described in Step 2 of Example 5, starting from(1r,4r)-methyl 4-(6-methoxyquinolin-2-yl)cyclohexanecarboxylate (72 mg,above product) to give the desired Compound IV-6. ¹H NMR (DMSO-d₆, 300MHz): δ 8.03 (1H, d, J=9 Hz), 7.75 (1H, d, J=9 hz), 7.33 (1H, d, J=9Hz), 7.24 (1H, dd, J=3, 9 Hz), 7.08 (1H, d, J=3 Hz), 3.30 (1H, m), 2.76(1H, m), 2.25-1.90 (4H, m) 1.75-1.50 (4H, m) ppm. MS (ESI): m/z 272.0[M+1]⁺.

Example 7 Compound IV-7:(1s,4s)-4-(6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid

Followed the procedure described in Step 2 of Example 5, starting from(1s,4s)-methyl 4-(6-methoxyquinolin-2-yl)cyclohexanecarboxylate (72 mg,see Example 6 Step 1 for synthesis) to give the desired Compound IV-7.¹H NMR (DMSO-d₆, 300 MHz): δ 7.97 (1H, d, J=9 Hz), 7.74 (1H, d, J=9 hz),7.26 (1H, dd, J=3, 9 Hz), 7.24 (1H, d, J=9 Hz), 7.08 (1H, d, J=3 Hz),3.34 (1H, m), 2.75 (1H, m), 2.25-1.50 (8H, m) ppm. MS (ESI): m/z 272.0[M+1]⁺.

Example 8 Compound IV-8: 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid

Followed Scheme 2, B Conditions

Step 1: Synthesis of 3-chloro-4-(6-methoxyquinolin-2-yl)benzoic acid

A mixture of 2-chloro-6-methoxyquinoline (Intermediate 1) (200 mg, 1.04mmol), 4-carboxy-2-chlorophenylboronic acid (247 mg, 1.24 mmol) andK₂CO₃ (369 mg, 2.70 mmol) in DEGME/H₂O (7.0 mL/2.0 mL) was degassedthree times under N₂ atmosphere. Then PdCl₂(dppf) (75 mg, 0.104 mmol)was added and the mixture was heated to 110° C. for 3 hours under N₂atmosphere. The reaction mixture was diluted with EtOAc (100 mL) andfiltered. The filtrate was washed with brine (20 mL), dried over Na₂SO₄,filtered and concentrated to give3-chloro-4-(6-methoxyquinolin-2-yl)benzoic acid (150 mg, yield 46%) as ayellow solid, which was used for the next step without furtherpurification.

Step 2: Synthesis of Compound IV-8

To a suspension of 3-chloro-4-(6-methoxyquinolin-2-yl)benzoic acid (150mg, 0.479 mmol) in anhydrous CH₂Cl₂ (5 mL) was added AlCl₃ (320 mg, 2.40mmol). The reaction mixture was refluxed overnight. The mixture wasquenched with saturated NH₄Cl (10 mL) and the aqueous layer wasextracted with CH₂Cl₂/MeOH (v/v=10:1, 30 mL×3). The combined organiclayer was washed with brine, dried over Na₂SO₄, filtered, andconcentrated to give the crude product, which was purified by prep-HPLC(0.1% TFA as additive) to give3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (25 mg, yield 18%). ¹HNMR (DMSO, 400 MHz): δ 10.20 (brs, 1H), 8.30 (d, J=8.4 Hz, 1H),8.10-8.00 (m, 2H), 7.95 (d, J=9.2 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.72(d, J=8.8 Hz, 1H), 7.38 (dd, J=6.4, 2.8 Hz, 1H), 7.22 (d, J=2.4 Hz, 1H),MS (ESI): m/z 299.9 [M+H]⁺.

Example 9 Compound IV-9: 2-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid

Followed Scheme 3

A mixture of 2-chloroquinolin-6-ol (Intermediate 2) (50 mg, 0.270 mmol),4-borono-2-chlorobenzoic acid (55 mg, 0.270 mmol), K₂CO₃ (75 mg, 0.540mmol) and Pd(dppf)Cl₂ (25 mg, 0.0306 mmol) in2-(2-methoxyethoxyl)ethanol (1.5 mL) and water (0.4 mL) was stirredunder N₂ atmosphere at 130° C. for 3 hours. The resulting mixture wascooled to room temperature and filtered. The filtrate was concentratedunder reduced pressure and the residue was purified by prep. HPLC (0.1%TFA as additive) to give Compound IV-9 (33 mg, yield 40%) as yellowsolid. ¹H NMR (CD₃OD, 400 MHz): δ 8.39 (d, J=8.4 Hz, 1H), 8.29 (d, J=1.6Hz, 1H), 8.16-8.02 (m, 4H), 7.47 (dd, J=9.2, 2.8 Hz, 1H), 7.25 (d, J=2.4Hz, 1H). MS (ESI): m/z 298.0 [M−1]⁻.

Example 10 Compound IV-10: 2-fluoro-4-(6-hydroxyquinolin-2-yl)benzoicacid

Followed Scheme 3: Starting Materials: 2-chloroquinolin-6-ol(Intermediate 2) and 4-borono-2-fluorobenzoic acid. ¹H NMR (CD₃OD, 400MHz): δ 8.54 (d, J=8.4 Hz, 1H), 8.20-8.06 (m, 3H), 8.06-7.96 (m, 2H),7.55 (dd, J=9.2, 2.4 Hz, 1H), 7.32 (d, J=2.8 Hz, 1H). MS (ESI): m/z283.6 [M+1]⁺.

Example 11 Compound IV-11:2-(4-(2H-tetrazol-5-yl)phenyl)-4-chloroquinolin-6-ol

Step 1: Synthesis of 4-(4-chloro-6-methoxyquinolin-2-yl)benzonitrile

Followed Scheme 2, Step 1 starting from 4-cyanophenylboronic acid and2,4-dichloro-6-methoxyquinoline, where the solvent used was DMF, and thecatalyst used was Pd(PPh₃)₄. The mixture was heated to 100° C. for 3 h,allowed to cool, and then poured into ice water. The resulting solid wasisolated by filtration, washed with water, and dried followed byrecrystallization from methanol to give the desired product.

Step 2: Synthesis of 4-(4-chloro-6-hydroxyquinolin-2-yl)benzonitrile

Followed the procedure for Scheme 1, step 2, with an EthylAcetate/aqueous workup. Purification by prep-TLC gave desired product.

Step 3: Synthesis of2-(4-(2H-tetrazol-5-yl)phenyl)-4-chloroquinolin-6-ol

Followed Scheme 4, route A. To a solution of4-(4-chloro-6-hydroxyquinolin-2-yl)benzonitrile (65 mg, 0.23 mmol) intoluene (2 mL), was added TMSN₃ (455 mg, 4.18 mmol) and Bu₂SnO (15 mg,0.069 mmol) at room temperature. The mixture was heated to refluxovernight. The volatiles were removed under reduced pressure. Theresidue was purified by prep-HPLC to afford Compound IV-11 (10 mg,13.5%). ¹H NMR (MeOD-d₄, 500 MHz): δ 8.34 (d, J=8.5 Hz, 2H), 8.21 (s,1H), 8.19 (s, 2H), 8.07 (d, J=9.0 Hz, 1H), 7.51 (d, J=2.5 Hz, 1H), 7.47(dd, J=2.5 Hz, J=9.5 Hz, 1H). MS (ESI): m/z 324.0 [M+1]⁺.

Example 12 Compound IV-12:3-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-5(2H)-one

Step 1: Synthesis of 4-(6-hydroxyquinolin-2-yl)benzonitrile

Followed Scheme 3 starting from 2-chloroquinolin-6-ol (Intermediate 2)and 4-cyanophenylboronic acid, and using 1,4-Dioxane:H₂O solution.Reaction was run at 100° C. in a microwave reactor for 1 hour. Ethylacetate workup was followed by column chromatography (5% to 50% EtOAc inhexanes gradient).

Step 2: Synthesis of N-hydroxy-4-(6-hydroxyquinolin-2-yl)benzimidamide

See Scheme 4, route B. 4-(6-hydroxyquinolin-2-yl)benzonitrile (900 mg,3.68 mmol) was dissolved in 25 mL of EtOH and to it was added NH₂OH.HCl(500 mg, 7.36 mmol) and TEA (1.5 mL). The mixture was stirred at 80° C.for 2 hours followed by concentration in vacuo. The crude solids werethen suspended in 25 mL of H₂O and stirred for 1 hour. Filtration anddrying of the solids yielded desired product (800 mg, 78% yield).

Step 3: Synthesis of(3-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-5(2H)-one)

N-hydroxy-4-(6-hydroxyquinolin-2-yl)benzimidamide (800 mg, 2.86 mmol)was dissolved in 25 mL of THF and CDI (557 mg, 3.44 mmol) and TEA (0.2mL) was added. The mixture was stirred at 65° C. for 2 hours, followedby concentration in vacuo. The crude material was dissolved in 10 mL 1NNaOH and filtered through Celite. The mixture was then acidified with 1NHCl to a pH of 4.5 and the solids were filtered and dried. The solidswere slurried in 10 mL of EtOAc overnight at 50° C. followed byfiltration to yield Compound IV-12 (315 mg, 36% yield). ¹H NMR (DMSO-d₆,400 MHz): δ 10.19 (s, 1H), 8.42 (d, 2H), 8.28 (d, 1H), 8.14-8.11 (d,1H), 8.00-7.94 (m, 3H), 7.39-7.35 (dd, 1H), 7.21 (s, 1H). MS (ESI): m/z306.44 [M+H]⁺.

Example 13 Compound IV-13: 3-fluoro-4-(6-hydroxyquinolin-2-yl)benzoicacid

Followed Scheme 3: Starting Materials: 2-chloroquinolin-6-ol and4-borono-3-fluorobenzoic acid. ¹H NMR (CD₃OD, 400 MHz): δ 8.31 (d, J=8.4Hz, 1H), 7.94-7.86 (m, 3H), 7.81-7.74 (m, 2H), 7.35 (dd, J=9.2, 2.8 Hz,1H), 7.15 (d, J=2.8 Hz, 1H). MS (ESI): m/z 283.6 [M+H]⁺.

Example 14 Compound IV-14: 4-(6-hydroxyquinolin-2-yl)-3-methoxybenzoicacid

Followed Scheme 3: Starting Materials: 2-chloroquinolin-6-ol and4-borono-3-methoxybenzoic acid. ¹H NMR (CD₃OD, 400 MHz): δ 8.81 (d,J=8.4 Hz, 1H), 8.14 (d, J=9.2 Hz, 1H), 8.10 (d, J=8.4 Hz, 1H), 7.89 (dd,J=6.8, 1.6 Hz, 2H), 7.85 (d, J=8.4 Hz, 1H), 7.69 (dd, J=9.2, 2.8 Hz,1H), 7.48 (d, J=2.8 Hz, 1H). MS (ESI): m/z 295.7 [M+H]⁺.

Example 15 Compound IV-15:5-(6-hydroxyquinolin-2-yl)thiophene-2-carboxylic acid

Followed Scheme 3: Starting Materials: 2-chloroquinolin-6-ol and5-boronothiophene-2-carboxylic acid. ¹H NMR (CD₃OD, 400 MHz): δ 8.25 (d,J=8.8 Hz, 1H), 7.97 (dd, J=9.2, 3.2 Hz, 2H), 7.88-7.82 (m, 2H), 7.41(dd, J=9.2, 2.8 Hz, 1H), 7.19 (d, J=2.4 Hz, 1H). MS (ESI): m/z 271.6[M+H]⁺.

Example 16 Compound IV-16:4-(6-hydroxyquinolin-2-yl)cyclohex-3-enecarboxylic acid

Step 1: Synthesis of methyl4-(6-hydroxyquinolin-2-yl)cyclohex-3-enecarboxylate

Followed Scheme 3: Starting Materials: 2-chloroquinolin-6-ol and methyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-enecarboxylate.

Step 2: Synthesis of 4-(6-hydroxyquinolin-2-yl)cyclohex-3-enecarboxylicacid

Basic hydrolysis conditions with LiOH gave the final desired product. ¹HNMR (DMSO-d₆, 300 MHz): δ 12.24 (1H, s), 9.92 (1H, s), 8.04 (1H, d, J=9Hz), 7.75 (1H, d, J=9 hz), 7.66 (1H, d, J=9 Hz), 7.24 (1H, dd, J=3, 9Hz), 7.08 (1H, d, J=3 Hz), 6.74 (1H, s), 3.30 (1H, m), 2.84 (1H, m),2.50 (4H, m), 2.08 (1H, m), 1.71 (1H, m) ppm. MS (ESI): m/z 270.0[M+1]⁺.

Example 17 Compound IV-17: 4-(3-fluoro-6-hydroxyquinolin-2-yl)benzoicacid

Step 1: Synthesis of 4-(4-chloro-3-fluoro-6-methoxyquinolin-2-yl)benzoicacid

Followed Scheme 3 where the starting materials were2,4-dichloro-3-fluoro-6-methoxyquinoline (Intermediate 3) and4-boronobenzoic acid where the crude was purified by silica gel column(PE/EtOAc=1/1) to give a mixture of compound4-(4-chloro-3-fluoro-6-methoxyquinolin-2-yl)benzoic acid and4-(3-fluoro-6-methoxyquinolin-2-yl)benzoic acid, which was used for thenext step without further purification.

Step 2: Synthesis of 4-(3-fluoro-6-methoxyquinolin-2-yl)benzoic acid

To a solution of the mixture from Step 1 in absolute MeOH (5 mL) wasadded Pd/C (10% Pd, 100 mg). The mixture was stirred at 25° C. for 1hour under H₂ atmosphere. The solids were filtered off and the filtratewas concentrated to give the product (60 mg, two-step yield 66%).

Step 3: Synthesis of 4-(3-fluoro-6-hydroxyquinolin-2-yl)benzoic acid

Followed Scheme 2, step 2, B conditions. ¹H NMR (DMSO, 400 MHz): δ 13.35(brs, 1H), 10.40 (brs, 1H), 8.25 (d, J=12.8 Hz, 1H), 8.27 (s, 4H), 8.01(d, J=9.2 Hz, 1H), 7.40 (dd, J=8.8, 2.4 Hz, 1H), 7.25 (d, J=2.8 Hz, 1H).

Example 18 Compound IV-18:4-(4-chloro-3-fluoro-6-hydroxyquinolin-2-yl)benzoic acid

Step 1: Synthesis of 4-(4-chloro-3-fluoro-6-methoxyquinolin-2-yl)benzoicacid

Synthesis described in Step 1 of Example 17.

Step 2: Synthesis of 4-(4-chloro-3-fluoro-6-hydroxyquinolin-2-yl)benzoicacid

Followed Scheme 2, Step 2, B conditions. ¹H NMR (DMSO, 400 MHz): δ 13.25(brs, 1H), 10.75 (brs, 1H), 8.10 (s, 4H), 8.05 (d, J=9.2 Hz, 1H), 7.41(dd, J=9.2, 2.8 Hz, 1H), 7.35 (d, J=2.4 Hz, 1H).

Example 19 Compound IV-19: 4-(3-chloro-6-hydroxyquinolin-2-yl)benzoicacid

Step 1: Synthesis of 4-(3-chloro-6-methoxyquinolin-2-yl)benzoic acid

Followed Scheme 3 where the starting materials were2,3-dichloro-6-methoxyquinoline (Intermediate 4) and 4-boronobenzoicacid.

Step 2: Synthesis of 4-(4-chloro-3-fluoro-6-hydroxyquinolin-2-yl)benzoicacid

Followed Scheme 2, Step 2, B conditions. ¹H NMR (CD₃OD, 400 MHz): δ 8.35(s, 1H), 8.17 (d, J=8.0 Hz, 2H), 7.95 (d, J=9.2 Hz, 1H), 7.80 (d, J=8.0Hz, 2H), 7.40 (dd, J=9.2, 2.4 Hz, 1H), 7.15 (d, J=2.8 Hz, 1H). MS (ESI):m/z 299.8 [M+H]⁺.

Example 20 Compound IV-20:3-(2-fluoro-4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-5(4H)-one

Step 1: Synthesis of 2-fluoro-4-(6-hydroxyquinolin-2-yl)benzonitrile

Followed Scheme 3 starting from 2-chloroquinolin-6-ol (Intermediate 2)and 4-cyano-3-fluorophenylboronic acid where crude was purified bysilica gel column chromatography.

Step 2 and 3: Synthesis of3-(2-fluoro-4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-5(4H)-one

Followed Scheme 4, route B, step 1: After solvent was removed in vacuo,the crude 2-fluoro-N-hydroxy-4-(6-hydroxyquinolin-2-yl)benzimidamide wastaken on without purification. Followed Scheme 4, route B, step 2:Purification by silica gel column, eluting with 10% MeOH in DCM gavedesired product. ¹H NMR (DMSO-d₆, 300 MHz): δ 10.22 (1H, s), 8.27-8.33(2H, m), 8.16 (1H, d, J=9 Hz), 7.91-7.99 (2H, m), 7.66 (1H, d, J=9 Hz),7.39 (1H, dd, J=3, 9 Hz), 7.21 (1H, d, J=3 Hz) ppm. MS (ESI): m/z 324.0[M+1]⁺.

Example 21 Compound IV-21:3-(3-fluoro-4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-5(4H)-one

Followed procedure described for Compound IV-20 in Example 20 startingfrom 2-chloroquinolin-6-ol (Intermediate 2) and4-cyano-2-fluorophenylboronic acid. ¹H NMR (DMSO-d₆, 300 MHz): δ 10.22(1H, s), 8.27-8.33 (2H, m), 7.97 (1H, d, J=9 Hz), 7.77-7.82 (2H, m),7.39 (1H, dd, J=3, 9 Hz), 7.21 (1H, d, J=3 Hz) ppm. MS (ESI): m/z 324.0[M+1]⁺.

Example 22 Compound IV-22: 4-(4-chloro-6-hydroxyquinolin-2-yl)benzoicacid

Step 1: Synthesis of 4-(4-chloro-6-hydroxyquinolin-2-yl)benzonitrile

Followed Step 2 of Scheme 1 starting from4-(4-chloro-6-methoxyquinolin-2-yl)benzonitrile (see step 1 of Example11 for synthesis).

Step 2: Synthesis of 4-(4-chloro-6-hydroxyquinolin-2-yl)benzoic acid

A solution of 4-(4-chloro-6-hydroxyquinolin-2-yl)benzonitrile (40 mg,0.14 mmol), con. HCl (1 mL) and dioxane (1 mL) was heated at 90° C.overnight. The mixture was extracted with ethyl acetate. The organiclayer was concentrated and purified by prep-HPLC to afford CompoundIV-22 (10 mg, yield: 23%). ¹H NMR (MeOD-d₄, 500 MHz): 8.16 (d, J=8.0 Hz,2H), 8.10 (d, J=8.5 Hz, 2H), 8.07 (s, 1H), 7.96 (d, J=9.0 Hz, 1H), 7.41(d, J=2.5 Hz, 1H), 7.35 (dd, J=3.0 Hz, J=9.0 Hz, 1H). MS (ESI): m/z 300[M+1]⁺.

Example 23 Compound IV-23:2-(2-chloro-4-(2H-tetrazol-5-yl)phenyl)quinolin-6-ol

Example procedure for Scheme 5, Compound C.

Step 1: Synthesis of 3-chloro-4-(6-hydroxyquinolin-2-yl)benzamide

A mixture of 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (CompoundIV-8, Example 8) (400 mg, 1.33 mmol) and SOCl₂ (10 mL) was refluxed for1 hour, then concentrated under reduced pressure to give crude product(400 mg) as off-white solid. To this solid was added NH₄OH (10 mL) andthe reaction mixture was stirred at 30° C. for 1 hour. The resultingmixture was acidified with aqueous HCl (2 M) until pH=6 and extractedwith EtOAc (50 mL×3), dried over Na₂SO₄ and concentrated to give crudeproduct (360 mg) as a solid.

Step 2: Synthesis of 3-chloro-4-(6-hydroxyquinolin-2-yl)benzonitrile

A mixture of crude 3-chloro-4-(6-hydroxyquinolin-2-yl)benzamide (360mg), TFAA (505 mg, 2.40 mmol) and Et₃N (364 mg, 3.62 mmol) in DCM (20mL) was stirred at 30° C. overnight. The resulting mixture was suspendedin water (50 mL) and extracted with EtOAc (50 mL x 3). The combinedorganic layers were dried over Na₂SO₄ and concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (PE/EtOAc=10/1) to give the product (250 mg, 3-step yield 67%) as asolid.

Step 3: Synthesis of2-(2-chloro-4-(2H-tetrazol-5-yl)phenyl)quinolin-6-ol

A mixture of 3-chloro-4-(6-hydroxyquinolin-2-yl)benzonitrile (230 mg,0.819 mmol), NaN₃ (55 mg, 0.820 mmol) and LiCl (70 mg, 1.64 mmol) indiethylene glycol monomethyl ether (5 mL) was refluxed for 4 hours. Theresulting mixture was cooled, filtered through silica gel pad andpurified by prep-HPLC (0.1% TFA as additive) to give Compound IV-23 (32mg, yield 12%). ¹H NMR (CD₃OD, 400 MHz): δ 8.52 (d, J=8.4 Hz, 1H), 8.34(s, 1H), 8.21 (d, J=8.0 Hz, 1H), 8.04 (d, J=9.2 Hz, 1H), 7.92-7.82 (m,2H), 7.54 (dd, J=9.2, 2.0 Hz, 1H), 7.34 (d, J=2.0 Hz, 1H). MS (ESI): m/z323.6 [M+H]⁺.

Example 24 Compound IV-24:5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,3,4-oxadiazol-2(3H)-one

Example procedure for Scheme 5, Compound B.

Step 1: Synthesis of tert-butyl 2-(4-(6-hydroxyquinolin-2-yl)benzoyl)hydrazinecarboxylate

A mixture of 4-(6-hydroxyquinolin-2-yl)benzoic acid (Compound IV-3,Example 3) (200 mg, 0.754 mmol), EDCI (145 mg, 0.754 mmol) and BocNHNH₂(100 mg, 0.754 mmol) in DCM (10 mL) and DMF (10 mL) was stirred at 25°C. overnight, followed by an aqueous/EtOAc workup. The crude waspurified by column chromatography on silica gel (PE/EtOAc=1/1) to givethe product (200 mg, yield 70%) as a yellow solid.

Step 2: Synthesis of 4-(6-hydroxyquinolin-2-yl)benzohydrazide

A mixture of tert-butyl2-(4-(6-hydroxyquinolin-2-yl)benzoyl)hydrazinecarboxylate (240 mg, 0.633mmol) and HCl/MeOH (20 mL, 4M) was stirred at 25° C. overnight. Theresulting mixture was concentrated under reduced pressure to dryness togive the product (120 mg, yield 68%).

Step 3: Synthesis of5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,3,4-oxadiazol-2(3H)-one

A mixture of 4-(6-hydroxyquinolin-2-yl)benzohydrazide (90 mg, 0.322mmol) and CDI (454 mg, 3.22 mmol) in DCM (20 mL) was refluxed overnight.The resulting mixture was cooled to room temperature, followed by anaqueous/EtOAc workup. The crude was purified by prep-HPLC (0.1% TFA asadditive) to give Compound IV-24 (18 mg, yield 11%). ¹H NMR (CD₃OD, 400MHz): δ 8.64-8.48 (m, 1H), 8.38-7.96 (m, 6H), 7.66-7.24 (m, 2H). MS(ESI): m/z 305.7 [M+H]⁺.

Example 25 Compound IV-25:3-(dimethylamino)-4-(6-hydroxyquinolin-2-yl)benzoic acid

Step 1: Synthesis of methyl 3-amino-4-(6-methoxyquinolin-2-yl)benzoate

Followed Scheme 2, step 1, B conditions starting from2-amino-4-(methoxycarbonyl)phenylboronic acid and2-chloro-6-methoxyquinoline, with a purification by columnchromatography on silica gel (PE/EtOAc=8/1) to give the product.

Step 2: Synthesis of methyl3-(dimethylamino)-4-(6-methoxyquinolin-2-yl)benzoate

To a solution of above compound (400 mg, 1.30 mmol) in MeOH/CH₂Cl₂ (10mL/20 mL) was added aqueous HCHO (37% in water, 0.4 mL), followed byNaBH₃CN (327 mg, 5.19 mmol) and ZnCl₂ (348 mg, 2.60 mmol) at 0° C. Themixture was stirred at 30° C. for 6 hours. The mixture was quenched withice water, the aqueous layer was extracted with CH₂Cl₂ (30 mL×3), thecombined organic layer was washed with brine (30 mL), dried overanhydrous Na₂SO₄ and concentrated in vacuo to give the product (400 mg,yield 92%).

Step 3: Synthesis of methyl3-(dimethylamino)-4-(6-hydroxyquinolin-2-yl)benzoate

Followed Scheme 2, step 2, B conditions where crude was purified bycolumn chromatography on silica gel (PE/EtOAc=5/1) to give the desiredproduct.

Step 4: Synthesis of 3-(dimethylamino)-4-(6-hydroxyquinolin-2-yl)benzoicacid

To a solution of above compound (290 mg, 0.901 mmol) in THF/MeOH (8 mL/4mL) was added aqueous LiOH (1 M, 4 mL). The mixture was stirred at 30°C. for 3 hours. The mixture was diluted with H₂O (10 mL), the aqueouslayer was neutralized by 2M HCl to pH 7. An aqueous/CH₂Cl₂ workup wasfollowed by column chromatography on silica gel (CH₂Cl₂/MeOH=10/1) togive Compound IV-25 (80 mg, yield 29%). ¹H NMR (MeOD-d₄, 400 MHz): δ8.15 (d, J=8.8 Hz, 1H), 7.95 (d, J=9.2 Hz, 1H), 7.87-7.81 (m, 2H), 7.76(d, J=8.0 Hz, 1H), 7.60 (d, J=8.0 Hz, 1H), 7.38 (dd, J=8.8, 2.4 Hz, 1H),7.20 (d, J=2.4 Hz, 1H), 2.65 (s, 6H). MS (ESI): m/z 308.9 [M+H]⁺.

Example 26 Compound IV-26: 4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoicacid

Step 1: Synthesis of 4-(4-fluoro-6-methoxyquinolin-2-yl)benzonitrile

A mixture of 4-(4-chloro-6-methoxyquinolin-2-yl)benzonitrile (SeeExample 11, step 1) (1 g, 3.4 mmol), CsF (5.2 g, 34 mmol), and n-Bu₄NBr(109 mg, 0.34 mmol) in DMSO (10 mL) was heated to 150° C. for 2 h. Anaqueous/EtOAC workup was followed by purification by columnchromatography (PE/EA=10/1) to afford desired product (300 mg, 31.7%).

Step 2: Synthesis of 4-(4-fluoro-6-hydroxyquinolin-2-yl)benzonitrile

Followed Step 2 of Scheme 1, where an aqueous/EtOAc workup was followedby column chromatography (PE:EA=4:1) to give the desired product.

Step 3: Synthesis of 4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid

A mixture of above (100 mg, 0.38 mmol) and NaOH (152 mg, 3.8 mmol) in1,4-dioxane/H₂O (1 mL/1 mL) was heated to reflux overnight. Thevolatiles were removed under reduced pressure. The residue was purifiedby prep-HPLC and then prep-TLC (EtOAc) to afford Compound IV-26 (10 mg,9.3%). ¹H NMR (MeOD-d₄, 500 MHz): 8.22-8.17 (m, 4H), 8.04 (d, J=9.5 Hz,1H), 7.79 (d, J=12.0 Hz, 1H), 7.43 (dd, J=2.5 Hz, J=9.0 Hz, 1H), 7.31(d, J=2.0 Hz, 1H). MS (ESI): m/z 284.0 [M+1]⁺.

Example 27 Compound IV-27: 4-(6-hydroxyquinolin-2-yl)-3-methylbenzoicacid

Followed Scheme 3: Starting Materials: 2-chloroquinolin-6-ol and methyl3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate. ¹H NMR(DMSO-d₆, 400 MHz): δ 10.45 (brs, 1H), 8.61-8.46 (m, 1H), 8.03 (d, J=8.8Hz, 1H), 7.96 (s, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.78 (d, J=8.8 Hz, 1H),7.66 (d, J=8.0 Hz, 1H), 7.50 (d, J=8.0 Hz, 1H), 7.35 (s, 1H), 2.42 (s,3H). MS (ESI): m/z 279.9 [M+H]⁺.

Example 28 Compound IV-28:4-(3-chloro-6-hydroxyquinolin-2-yl)-3-fluorobenzoic acid

Step 1: Synthesis of 4-(3-chloro-6-methoxyquinolin-2-yl)-3-fluorobenzoicacid

Scheme 3: starting from 2,3-dichloro-6-methoxyquinoline (Intermediate 4)and 4-borono-3-fluorobenzoic acid. An aqueous/EtOAc workup was performedfollowed by purification by silica gel column (PE/EtOAc=1/1) to give thedesired product.

Step 2: Synthesis of 4-(3-chloro-6-hydroxyquinolin-2-yl)-3-fluorobenzoicacid

Scheme 1, step 2: where reaction was refluxed for 18 hours, followed byan aqueous/EtOAc with 10% MeOH workup. Purification by prep-HPLC gaveCompound IV-28. ¹H NMR (DMSO-d₆, 400 MHz): 13.45 (brs, 1H), 10.39 (brs,1H), 8.52 (s, 1H), 7.94-7.88 (m, 2H), 7.80 (d, J=10.4 Hz, 1H), 7.67 (t,J=7.6 Hz, 1H), 7.38 (dd, J=6.8, 2.4 Hz, 1H), 7.21 (d, J=2.8 Hz, 1H). MS(ESI): m/z 317.8 [M+H]⁺.

Example 29 Compound IV-29:3-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-thiadiazol-5(2H)-one

Step 1: Synthesis of N-hydroxy-4-(6-methoxyquinolin-2-yl)benzimidamide

Scheme 4, route B, step 1: 4-(6-methoxyquinolin-2-yl)benzonitrile(prepared via scheme 3) (780 mg, 3.00 mmol) was suspended in methanol(10 mL) and hydroxylamine hydrochloride (639 mg, 10.7 mmol), K₂CO₃ (414mg, 3.00 mmol) were added. The reaction mixture was refluxed for 12hours. Water (15 mL) was added and the precipitated solid was collectedby filtration, washed with ethanol (5 mL) and dried over high vacuum togive the product (600 mg).

Step 2: Synthesis of3-(4-(6-methoxyquinolin-2-yl)phenyl)-1,2,4-thiadiazol-5(2H)-one

Scheme 4, route B, step 2: To a solution of above product (450 mg) inTHF (10 mL) was added TCDI (410 mg, 2.30 mmol) and the mixture wasstirred at 30° C. for 3 hours. After completion of the reaction, thesolvent was removed to give the product (300 mg).

Step 3: Synthesis of3-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-thiadiazol-5(2H)-one

Scheme 1, step 2: Reaction was refluxed overnight, followed by anaqueous/EtOAc workup and purification by prep-HPLC (0.1% TFA asadditive) to give Compound IV-29. ¹H NMR (DMSO-d₆, 300 MHz): 13.50 (brs,1H), 8.40-8.26 (m, 3H), 8.18-8.04 (m, 3H), 7.95 (d, J=9.0 Hz, 1H), 7.36(dd, J=9.0, 2.4 Hz, 1H), 7.20 (d, J=2.7 Hz, 1H). MS (ESI): m/z 322.0[M+H]⁺.

Example 30 Compound IV-30:4-(6-hydroxyquinolin-2-yl)-3-(trifluoromethyl)benzoic acid

Followed Scheme 3: starting with 2-chloroquinolin-6-ol and methyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)benzoate(Intermediate 5).

¹H NMR (DMSO-d₆, 400 MHz): δ 10.21 (brs, 1H), 8.35-8.25 (m, 3H), 7.89(d, J=9.2 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.38(dd, J=9.2, 2.8 Hz, 1H), 7.23 (d, J=2.4 Hz, 1H). MS (ESI): m/z 333.7[M+H]⁺.

Example 31 Compound IV-31:4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)benzoic acid

Followed Scheme 3: starting with 4-carboxyphenylboronic acid and2-chloro-3-(trifluoromethyl)quinolin-6-ol (Intermediate 6). ¹H NMR(DMSO, 400 MHz): δ 13.10 (brs, 1H), 10.45 (brs, 1H), 8.84 (s, 1H), 8.02(d, J=8.4 Hz, 2H), 7.97 (d, J=8.8 Hz, 1H), 7.60 (d, J=8.0 Hz, 2H), 7.50(dd, J=9.2, 2.8 Hz, 1H), 7.40 (d, J=2.8 Hz, 1H). MS (ESI): m/z 333.9[M+H]⁺.

Example 32 Compound IV-32: 2-(4-carboxyphenyl)-6-hydroxyquinoline1-oxide

Step 1: Synthesis of methyl 4-(6-hydroxyquinolin-2-yl)benzoate

Scheme 1, step 1, starting from 4-(methoxycarbonyl)phenylboronic acidand 2-chloroquinolin-6-ol, using Na₂CO₃ instead of TEA.

Step 2: Synthesis of 6-hydroxy-2-(4-(methoxycarbonyl)phenyl)quinoline1-oxide

To a solution of above compound (200 mg, 0.72 mmol) in 1,4-dioxane (5mL) was added mCPBA (372 mg, 2.16 mmol). The mixture was stirred at roomtemperature for three days. The volatiles were removed under reducedpressure. The residue was partitioned with sat. Na₂CO₃ solution (10 mL)and EtOAc (20 mL). The organic phase was washed with brine (10 mL),dried over Na₂SO₄, concentrated and purified by column chromatography toafford product (40 mg, 18.9%).

Step 3: Synthesis of 2-(4-carboxyphenyl)-6-hydroxyquinoline 1-oxide

A solution of the above compound (40 mg, 0.14 mmol) and NaOH (16 mg,0.41 mmol) in 1,4-dioxane/water (1.5 mL/0.5 mL) was heated at 80° C. for2 h. The volatiles were removed in vacuo. The residue was partitionedwith water (8 mL) and EtOAc (10 mL). The aqueous phase was separated,acidified with 1 N HCl to pH=5. The resulting precipitate was filtered,washed with water and EtOH, and dried in vacuo to afford Compound IV-32(10 mg, 25.6%).

¹H NMR (MeOD-d₄, 500 MHz): 8.61 (d, J=9.5 Hz, 1H), 8.20 (d, J=8.0 Hz,2H), 8.04-7.99 (m, 3H), 7.65 (d, J=8.5 Hz, 1H), 7.48 (dd, J=2.0 Hz,J=9.0 Hz, 1H), 7.30 (d, J=2.0 Hz, 1H); MS (ESI): m/z 282.0 [M+1]⁺.

Example 33 Compound IV-33:5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,3,4-thiadiazol-2(3H)-one

Example procedure for Scheme 5, Compound A:

Step 1: Synthesis of tert-butyl2-(4-(6-methoxyquinolin-2-yl)benzoyl)hydrazinecarboxylate

Scheme 5, step 1 of route A (see Example 24, step 1) starting from4-(6-methoxyquinolin-2-yl)benzoic acid (prepared following Scheme 3).

Step 2: Synthesis of tert-butyl2-(4-(6-methoxyquinolin-2-yl)phenylcarbonothioyl) hydrazinecarboxylate

A mixture of above compound (2.30 g, 5.85 mmol) and lawesson's reagent(2.30 g, 5.69 mmol) in anhydrous toluene (150 mL) was refluxedovernight. The resulting mixture was concentrated in vacuo. The residuewas washed with MeOH and the solid was isolated. The filtrate wasconcentrated in vacuo and the residue was purified by silica gel column(PE/EtOAc=3/1) to give crude tert-butyl2-(4-(6-methoxyquinolin-2-yl)phenylcarbonothioyl) hydrazinecarboxylate(2.00 g).

Step 3: Synthesis of5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,3,4-thiadiazol-2(3H)-one

A mixture of tert-butyl2-(4-(6-methoxyquinolin-2-yl)phenylcarbonothioyl) hydrazinecarboxylate(500 mg, 1.49 mmol) and AlCl₃ (600 mg, 4.50 mmol) in anhydrous DCM (50mL) was refluxed overnight. Reaction was cooled, followed by anaqueous/EtOAc workup. The residue was purified by prep-HPLC (0.1% TFA asadditive) to give Compound IV-33 (35 mg, yield 7%) as a solid. ¹H NMR(DMSO-d₆, 400 MHz): δ 13.22 (brs, 1H), 10.22 (brs, 1H), 8.40-8.28 (m,3H), 8.11 (d, J=8.8 Hz, 1H), 7.98 (d, J=8.8 Hz, 1H), 7.86 (d, J=8.4 Hz,2H), 7.38 (dd, J=9.2, 2.8 Hz, 1H), 7.22 (d, J=2.8 Hz, 1H). MS (ESI): m/z321.7 [M+H]⁺.

Example 34 Compound IV-34:5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-3(2H)-one

Step 1: Synthesis of 4-(6-methoxyquinolin-2-yl)benzamide

To a suspension of 4-(6-methoxyquinolin-2-yl)benzonitrile (5.00 g, 15.4mmol, prepared following Scheme 2, Step 1 starting from Intermediate 1and 4-cyanophenylboronic acid) in DMSO (40 mL) was added aqueous NaOH(1M, 10 mL). The mixture was cooled to 0° C. The aqueous H₂O₂ (30%, 30mL) was added dropwise. After addition, the mixture was stirred at 0° C.for 30 minutes. The mixture was quenched with saturated Na₂SO₃ (100 mL)and filtered. The precipitate was washed with H₂O (50 mL) and MeOH (50mL). The filter cake was dried via high vacuum to give the desiredproduct (5.50 g, yield: 99+%) as a solid.

Step 2: Synthesis of5-(4-(6-methoxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-3-ol

To a suspension of 4-(6-methoxyquinolin-2-yl)benzamide (2.00 g, 7.19mmol) in DCE (20 mL) was added oxalyl chloride (1.10 g, 8.99 mmol)rapidly at 30° C. The mixture was heated to 70° C. for 16 hours. Thesolvent was removed in vacuo to give a yellow solid (2.00 g), which wasused directly without further purification. A suspension of thismaterial (2.00 g) in TMSN₃ (30 mL) was heated to 90° C. for two days.The excess TMSN₃ was removed in vacuo and the residue was diluted withEtOH (200 mL). The mixture was filtered off and the filtrate wasconcentrated to give5-(4-(6-methoxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-3-ol (700 mg, twostep yield: 30%) as a solid.

Step 3: Synthesis of5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-oxadiazol-3-ol

Followed Scheme 2, Step 2, B conditions to give Compound IV-34 (60 mg,yield: 18%) as a solid. ¹H NMR (DMSO 400 MHz): δ 12.90 (brs, 1H), 10.15(brs, 1H), 8.45 (d, J=8.4 Hz, 2H), 8.30 (d, J=8.4 Hz, 1H), 8.15-8.10 (m,3H), 7.92 (d, J=9.2 Hz, 1H), 7.5 (dd, J=8.8, 2.4 Hz, 1H), 7.20 (d, J=2.8Hz, 1H). MS (ESI): m/z 305.9 [M+H]⁺.

Example 35 Compound IV-35:(1r,4r)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid

Step 1: Synthesis of ethyl4-(3-chloro-6-methoxyquinolin-2-yl)cyclohex-3-enecarboxylate

Followed Scheme 2, Step 1 (see Example 8, Step 1) starting from ethyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-enecarboxylateand 2,3-dichloro-6-methoxyquinoline (Intermediate 4).

Step 2: Synthesis of ethyl4-(3-chloro-6-methoxyquinolin-2-yl)cyclohexanecarboxylate

To ethyl 4-(3-chloro-6-methoxyquinolin-2-yl)cyclohex-3-enecarboxylate(900 mg, 2.60 mmol) in EtOH (40 mL) was added Rh(PPh₃)₃Cl (90.0 mg0.0900 mmol). The mixture was stirred at 30° C. for 4 days under H₂ (15psi). The mixture was filtered off and the filtrate was concentrated togive the crude product, which was purified by silica gel column(PE/EtOAc=10/1) to give the desired product (234 mg, yield 26%).

Step 3: Synthesis of(1r,4r)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid

To a mixture of ethyl4-(3-chloro-6-methoxyquinolin-2-yl)cyclohexanecarboxylate (200 mg, 0.580mmol) in anhydrous CH₂Cl₂ (10 mL) was added BBr₃ (0.3 mL, 2.9 mmol)dropwise at 0° C. The mixture was stirred at 0° C. for 2 hours. Water(10 mL) was added and the mixture was extracted with EtOAc (30 mL×3).The combined organic layers were washed with brine (20 mL×2), dried overNa₂SO₄, filtered and concentrated to give the crude product, which waspurified by prep-HPLC (0.1% TFA as additive) to give(1r,4r)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid(Compound IV-35, 65 mg, yield 37%) as a solid, and(1s,4s)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid(Compound IV-36, 26 mg, yield 15%) as a solid. The structure wasconfirmed by NOE. Data for Compound IV-35: ¹H NMR (DMSO 400 MHz): δ10.08 (brs, 1H), 8.27 (s, 1H), 7.79 (d, J=8.8 Hz, 1H), 7.26 (dd, J=9.2,2.8 Hz, 1H), 7.08 (d, J=2.8 Hz, 1H), 3.18 (tt, Jaa=12.0 Hz, Jea=3.2 Hz,1H), 2.28 (tt, Jaa=12.0 Hz, Jea=3.2 Hz, 1H), 2.03 (d, J=10.4 Hz, 2H),1.91 (d, J=10.8 Hz, 2H), 1.72-1.60 (m, 2H), 1.55-1.40 (m, 2H). MS (ESI):m/z 306.0 [M+H]⁺.

Example 36 Compound IV-36:(1s,4s)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid

See Example 35 for synthesis. ¹H NMR (DMSO 400 MHz): δ 10.11 (brs, 1H),8.28 (s, 1H), 7.82 (d, J=9.2 Hz, 1H), 7.27 (dd, J=9.2, 2.8 Hz, 1H), 7.09(d, J=2.8 Hz, 1H), 3.32-3.19 (m, 1H), 2.70-2.60 (m, 1H), 2.25-2.14 (m,2H), 1.85-1.70 (m, 4H), 1.70-1.58 (m, 2H). MS (ESI): m/z 306.0 [M+H]⁺.

Example 37 Compound IV-37:3-chloro-4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid

Followed Scheme 2, B conditions starting with2-chloro-4-fluoro-6-methoxyquinoline (Intermediate 7) and4-carboxy-2-chlorophenylboronic acid. ¹H NMR (MeOD 400 MHz): δ 8.20 (d,J=1.2 Hz, 1H), 8.10 (dd, J=8.0, 1.6 Hz, 1H), 8.02 (d, J=9.2, 1.2 Hz,1H), 7.75 (d, J=8.0 Hz, 1H), 7.52-7.45 (m, 2H), 7.47 (d, J=2.4 Hz, 1H).MS (ESI): m/z 317.9 [M+H]⁺.

Example 38 Compound IV-38:2-(5-(2H-tetrazol-5-yl)thiophen-2-yl)quinolin-6-ol

Step 1 and 2: Synthesis of5-(6-hydroxyquinolin-2-yl)thiophene-2-carbonitrile

Followed the two step synthesis shown in Scheme 1 starting with2-chloro-6-methoxyquinoline and 5-cyanothiophen-2-ylboronic acid, withminor variations: the base used in step 1 was sodium carbonate and thesolvent was DME (dimethoxyethane)/water. In step 2, after quenchingw/ice water, a standard ethyl acetate extraction was preformed followedby purification by prep-TLC (PE: EA=1:1).

Step 3: Synthesis of 2-(5-(2H-tetrazol-5-yl)thiophen-2-yl)quinolin-6-ol

Followed Scheme 4, route A. ¹H-NMR (DMSO-d₆500 MHz): 10.15 (s, 1H), 8.23(d, J=9.0 Hz, 1H), 8.05 (d, J=8.5 Hz, 1H), 7.99 (d, J=4.0 Hz, 1H), 7.86(d, J=9.0 Hz, 1H), 7.78 (d, J=3.5 Hz, 1H), 7.34 (dd, J=2.5, 9.0 Hz, 1H),7.17 (d, J=2.0 Hz, 1H). MS (ESI): m/z 296.0 [M+1]⁺.

Example 39 Compound IV-39:5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-thiadiazol-3(2H)-one

Step 1: Synthesis of 4-(6-methoxyquinolin-2-yl)benzothioamide

To a suspension of 4-(6-methoxyquinolin-2-yl)benzamide (see Example 34step 1 for synthesis, 3.00 g, 10.8 mmol) in anhydrous toluene (50 mL)was added Lawessen Reagent (2.60 g, 6.44 mmol). The mixture was refluxedfor 3 hours. The mixture was diluted with MeOH (50 mL) and filtered off.The filtrate was concentrated under reduce pressure to give the crudeproduct, which was purified by silica gel column (PE/EtOAc=1/1) to givethe product (1.30 g, yield 41%) as a solid.

Step 2 and 3: Synthesis of5-(4-(6-methoxyquinolin-2-yl)phenyl)-1,2,4-thiadiazol-3(2H)-one

To a solution of 4-(6-methoxyquinolin-2-yl)benzothioamide (500 mg, 1.07mmol) in DCE (10 mL) was added oxalylchloride (0.3 mL, 3.9 mmol)dropwise at 0° C. The mixture was heated to 90° C. for 2 hours. TMSN₃(0.8 mL, 5.7 mmol) was added dropwise. The mixture was stirred at 100°C. for 2 hours. Water (10 mL) was added to the mixture. The mixture wasfiltered and the filter cake was washed with IPA (20 mL) to product (280mg, yield 49%).

Step 4: Synthesis of5-(4-(6-hydroxyquinolin-2-yl)phenyl)-1,2,4-thiadiazol-3(2H)-one

Followed Scheme 2, Step 2, B conditions for demethylation. ¹H NMR (DMSO400 MHz): δ 12.76 (brs, 1H), 10.16 (brs, 1H), 8.41 (d, J=8.4 Hz, 2H),8.29 (d, J=8.8 Hz, 1H), 8.15-8.02 (m, 3H), 7.95 (d, J=9.2 Hz, 1H), 7.36(dd, J=9.2, 2.8 Hz, 1H), 7.19 (d, J=2.4 Hz, 1H), MS (ESI): m/z 321.9[M+H]⁺.

Example 40 Compound IV-40:3-fluoro-4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid

The compound was prepared following Scheme 2, B conditions starting from2-chloro-4-fluoro-6-methoxyquinoline (Intermediate 7) and4-carboxy-2-fluorophenylboronic acid. Note: In step 2, afterpurification by prep HPLC, the collected fractions were immediatelyneutralized with sat.NaHCO₃ to pH 6, followed by extraction withEtOAc/MeOH (v/v=10/1, 50 mL×3). Combined organics were washed with brine(50 mL), dried over Na₂SO₄ and concentrated in vacuo to give the desiredproduct. ¹H NMR (MeOD 400 MHz): δ 8.10-8.00 (m, 2H), 7.98 (dd, J=8.0,1.2 Hz, 1H), 7.85 (dd, J=11.6, 1.2 Hz, 1H), 7.62 (dd, J=11.6, 2.0 Hz,1H), 7.45 (dd, J=9.2, 2.8 Hz, 1H), 7.31 (d, J=2.8 Hz, 1H). MS (ESI): m/z301.8 [M+H]⁺.

Example 41 Compound IV-41:1-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)piperidine-4-carboxylicacid

Step 1: Synthesis of methyl1-(6-methoxy-3-(trifluoromethyl)quinolin-2-yl)piperidine-4-carboxylate

To a solution of 2-chloro-6-methoxy-3-(trifluoromethyl)quinoline (1.00g, 3.85 mmol) (see Intermediate 6, Step 8 for synthesis) in IPA (5 mL)was added methyl-4-piperidinecarboxylate (5.50 g, 38.5 mmol) and Et₃N(1.17 g, 11.6 mmol). The mixture was heated to reflux for 48 hours. Themixture was diluted with water (100 mL) and extracted with DCM (100mL×3), the combined organic layers were washed with brine (200 mL),dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to give the crude product, which was purified by columnchromatography on silica gel (PE/EtOAc=15/1) to give the product (600mg, yield 43%) as a solid.

Step 2: Synthesis of1-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)piperidine-4-carboxylicacid

To a solution of methyl1-(6-methoxy-3-(trifluoromethyl)quinolin-2-yl)piperidine-4-carboxylate(300 mg, 0.815 mmol) in DCM (5 mL) was added BBr₃ (0.4 mL, 4.08 mmol)dropwise at 0° C. The mixture was stirred at 0° C. for 20 hours. Water(10 mL) was added to the mixture dropwise at 0° C. The mixture wasconcentrated in vacuo, then diluted with EtOAc (150 mL), filtered andconcentrated in vacuo. Purification by prep-HPLC (0.1% TFA as additive)to give Compound IV-41 (26 mg, yield 9.4%) as a solid. ¹H NMR (MeOD 400MHz): δ 8.51 (s, 1H), 7.82 (d, J=8.8 Hz, 1H), 7.41 (dd, J=8.8, 2.8 Hz,1H), 7.21 (d, J=2.8 Hz, 1H), 3.55-3.49 (m, 2H), 3.13-3.02 (m, 2H),2.58-2.49 (m, 1H), 2.09-2.00 (m, 2H), 1.93-1.82 (m, 2H). MS (ESI): m/z340.7 [M+H]⁺.

Example 42 Compound IV-42: 4-(5-chloro-6-hydroxyquinolin-2-yl)benzoicacid

Step 1: Synthesis of methyl 4-(6-methoxyquinolin-2-yl)benzoate

A mixture of 2-chloro-6-methoxyquinoline (Intermediate 1, 200 mg, 1.0mmol), 4-(methoxycarbonyl) phenylboronic acid (205 mg, 1.1 mmol),Pd(dppf)Cl₂ (366 mg, 0.5 mmol) and sodium carbonate (212 mg, 2.0 mmol)in 1,4-dioxane/water (3 mL/0.6 mL) was heated to 120° C. by microwavefor 1 h. The precipitates were filtered; washed with EA (10 mL), acetone(10 mL) and water (10 mL) separately; dried to afford product (120 mg,40.9%).

Step 2: Synthesis of methyl 4-(5-chloro-6-methoxyquinolin-2-yl)benzoate

To a solution of above (100 mg, 0.34 mmol) in AcOH (2 mL) was addedSO₂Cl₂ (55 mg, 0.41 mmol). The reaction mixture was heated to 60° C. for3 h. Then the mixture was stirred at room temperature overnight. Theprecipitates were filtrated, washed with AcOH (10 mL×3) and dried togive the product as a powder (100 mg, 90.1%).

Step 3: Synthesis of 4-(5-chloro-6-hydroxyquinolin-2-yl)benzoic acid

Followed Scheme 1, step 2, with a purification by prep HPLC to give theproduct. ¹H-NMR (DMSO-d₆ 500 MHz): 8.50 (d, J=9.0 Hz, 1H), 8.32 (d,J=8.0 Hz, 2H), 8.25 (d, J=8.5 Hz, 1H), 8.10 (d, J=8.0 Hz, 2H), 7.96 (d,J=9.5 Hz, 1H), 7.62 (d, J=9.0 Hz, 1H); MS (ESI): m/z 300.0 [M+1]⁺.

Example 43 Compound IV-43:(1r,4r)-4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylicacid

Step 1: Synthesis of ethyl4-(6-methoxy-3-(trifluoromethyl)quinolin-2-yl)cyclohex-3-enecarboxylate

Followed Scheme 2, Step 1 (see Example 8, Step 1) starting from ethyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-enecarboxylateand 2-chloro-6-methoxy-3-(trifluoromethyl)quinoline (see Intermediate 6,Step 8 for synthesis), with a purification by column chromatography(PE/EtOAc=10/1).

Step 2: Synthesis of ethyl4-(6-methoxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylate

Followed step 2 of Compound W-35 with 0.15 equiv of catalyst at 20° C.for 4 days under H₂ (15 psi).

Step 3: Synthesis of(1r,4r)-4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylicacid

Followed step 3 of Compound IV-35, where the reaction was stirred at 10°C. for 20 hours before quenching with water. Purified crude by prep-HPLCto give ethyl4-(6-methoxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylate(Compound W-43, 109 mg, yield 29%) as a solid, and(1s,4s)-4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylicacid (Compound IV-44, 25 mg, yield 7%) as a solid. Compound IV-43 Data:¹H NMR (DMSO 400 MHz): δ 10.27 (brs, 1H), 8.61 (s, 1H), 7.89 (d, J=9.2Hz, 1H), 7.44 (dd, J=9.2, 2.8 Hz, 1H), 7.32 (d, J=2.8 Hz, 1H), 3.01-2.91(m, 1H), 2.40-2.30 (m, 1H), 2.00-1.90 (m, 2H), 1.95-1.78 (m, 4H),1.53-1.38 (m, 2H). MS (ESI): m/z 339.9 [M+H]⁺.

Example 44 Compound IV-44:(1s,4s)-4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylicacid

See Example 43 for synthesis. ¹H NMR (DMSO 400 MHz): δ 10.26 (br s, 1H),8.59 (s, 1H), 7.89 (d, J=9.2 Hz, 1H), 7.43 (dd, J=9.2, 2.8 Hz, 1H), 7.31(d, J=2.0 Hz, 1H), 3.09-2.95 (m, 1H), 2.75-2.62 (m, 1H), 2.28-2.16 (m,2H), 1.98-1.80 (m, 2H), 1.60-1.55 (m, 4H). MS (ESI): m/z 339.9 [M+H]⁺.

Example 45 Compound V-1: 4-(6-hydroxyquinazolin-2-yl)benzoic acid

Followed Scheme 6, A conditions.

Step 1: Synthesis of 4-(6-methoxyquinazolin-2-yl)benzoic acid

A mixture of 2-chloro-6-methoxyquinazoline (Intermediate 8, 200 mg, 1.03mmol), 4-carboxyphenylboronic acid (205 mg, 1.23 mmol) and K₂CO₃ (370mg, 2.68 mmol) in DEGME/H₂O (7.0 mL/2.0 mL) was degassed three timesunder N₂ atmosphere. Then PdCl₂(dppf) (75 mg, 0.103 mmol) was added andthe mixture was heated to 100° C. for 3 hours under N₂ atmosphere. Thereaction mixture was diluted with H₂O (10 mL). The aqueous layerextracted with EtOAc/MeOH (v/v=10:1, 30 mL×2). Then the aqueous layerwas acidified with 2M HCl to pH 6. The precipitate was collected byfiltration and washed with MeOH (10 mL), dried under high vacuum to givethe desired product (220 mg, yield 76%). ¹H NMR (DMSO 400 MHz): δ 13.10(brs, 1H), 9.63 (s, 1H), 8.62 (d, J=8.0 Hz, 2H), 8.10 (d, J=8.0 Hz, 2H),8.05 (d, J=9.2 Hz, 1H), 7.70 (dd, J=9.2, 2.8 Hz, 1H), 7.60 (d, J=2.4 Hz,1H), 3.98 (s, 3H).

Step 2: Synthesis of 4-(6-hydroxyquinazolin-2-yl)benzoic acid

To a suspension of 4-(6-methoxyquinazolin-2-yl)benzoic acid (220 mg,0.785 mmol) in anhydrous CH₂Cl₂ (5 mL) was added AlCl₃ (600 mg, 4.55mmol), the reaction mixture was refluxed overnight. The mixture wasquenched with sat.NH₄Cl (10 mL), the aqueous layer was extracted withCH₂Cl₂/MeOH (v/v=10/1, 30 mL×3), the combined organic layer was washedwith brine (30 mL), dried over Na₂SO₄, filtered and concentrated to givethe crude product, which purified by prep-HPLC (0.1% TFA as additive) togive Compound V-1 (27 mg, yield 13%). ¹H NMR (MeOD 400 MHz): δ 9.42 (s,1H), 8.60 (d, J=8.4 Hz, 2H), 8.17 (d, J=8.4 Hz, 2H), 8.00 (d, J=9.2 Hz,1H), 7.60 (dd, J=9.2, 2.8 Hz, 1H), 7.30 (d, J=2.8 Hz, 1H). MS (ESI): m/z266.9 [M+H]⁺.

Example 46 Compound V-2: 4-(6-hydroxy-4-methylquinazolin-2-yl)benzoicacid

Followed Scheme 6, B conditions.

Step 1: Synthesis of 4-(6-methoxy-4-methylquinazolin-2-yl)benzoic acid

To a mixture of 2-chloro-6-methoxy-4-methylquinazoline (Intermediate 9,350 mg, 1.68 mmol), 4-carboxyphenylboronic acid (418 mg, 2.52 mmol), andK₂CO₃ (464 mg, 3.36 mmol) in DEGME/H₂O (7 mL/1 mL) was added Pd(dppf)Cl₂(30.2 mg, 0.0369 mmol) under N₂ atmosphere. Then the mixture was heatedto 120° C. for 2 hours. To the reaction mixture was added H₂O (30 mL),extracted with EtOAc (15 mL×2), the aqueous layer was acidified with 1MHCl to pH=1-2, filtered and the filter cake was washed with MeOH anddried under high vacuum to give the product (380 mg, yield 78%). ¹H NMR(DMSO-d₆ 400 MHz): δ 8.61 (d, J=8.0 Hz, 2H), 8.10 (d, J=8.4 Hz, 2H),7.99 (d, J=9.2 Hz, 1H), 7.65 (dd, J=9.2, 2.8 Hz, 1H), 7.54 (d, J=2.8 Hz,1H), 3.98 (s, 3H), 2.99 (s, 3H).

Step 2: Synthesis of 4-(6-hydroxy-4-methylquinazolin-2-yl)benzoic acid

To a solution of 4-(6-methoxy-4-methylquinazolin-2-yl)benzoic acid (180mg, 0.612 mmol) in anhydrous DCM (10 mL) was added BBr₃ (0.29 mL, 3.05mmol, d=2.64 g/mL) dropwise at 0° C. The resulting mixture was stirredat 25° C. for 16 hours. The reaction mixture was quenched with H₂O (30mL), then extracted with DCM (20 mL×3). The combined organic layers werewashed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated to give the crude product. The crude product was washedwith MeOH (10 mL) to give Compound V-2 (130 mg, yield 76%). ¹H NMR(DMSO-d₆ 400 MHz): δ 13.06 (brs, 1H), 10.46 (brs, 1H), 8.60 (d, J=8.4Hz, 2H), 8.09 (d, J=8.4 Hz, 2H), 7.95 (d, J=9.2 Hz, 1H), 7.56 (dd,J=8.8, 2.4 Hz, 1H), 7.41 (d, J=2.4 Hz, 1H), 2.90 (s, 3H). MS (ESI): m/z280.9 [M+H]⁺.

Example 47 Compound V-3: 3-chloro-4-(6-hydroxyquinazolin-2-yl)benzoicacid

Prepared according to Scheme 6, B conditions, starting from Intermediate8 and 4-carboxy-2-chlorophenylboronic acid. ¹H NMR (DMSO-d₆ 400 MHz): δ10.60 (brs, 1H), 9.56 (s, 1H), 8.10-7.89 (m, 4H), 7.62 (dd, J=9.2, 2.8Hz, 1H), 7.38 (d, J=2.8 Hz, 1H). MS (ESI): m/z 300.8 [M+H]⁺.

Example 48 Compound V-4: 4-(6-hydroxyquinazolin-2-yl)-3-methylbenzoicacid

Prepared according to Scheme 6, B conditions, starting from Intermediate8 and methyl3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate. ¹H NMR(DMSO-d₆ 400 MHz): δ 9.54 (s, 1H), 8.00-7.86 (m, 4H), 7.60 (dd, J=8.8,2.4 Hz, 1H), 7.36 (d, J=2.4 Hz, 1H), 2.59 (s, 3H). MS (ESI): m/z280.9[M+H]⁺.

Example 49 Compound V-5: 4-(6-hydroxyquinazolin-2-yl)-3-methoxybenzoicacid

Prepared according to the first step of Compound V-2 starting fromIntermediate 10 and 4-borono-3-methoxybenzoic acid. ¹H NMR (DMSO-d₆ 400MHz): δ 9.53 (s, 1H), 7.94 (d, J=9.2 Hz, 1H), 7.72 (d, J=8.4 Hz, 1H),7.70-7.58 (m, 3H), 7.37 (d, J=2.8 Hz, 1H), 3.84 (s, 3H). MS (ESI): m/z296.9 [M+H]⁺.

Example 50 Compound V-6: 3-fluoro-4-(6-hydroxyquinazolin-2-yl)benzoicacid

Prepared according to Scheme 6, B conditions starting from Intermediate8 and 4-carboxy-2-fluorophenylboronic acid, where the crude product ofstep 2 was purified by prep-HPLC (0.1% TFA as additive) after which mostof the CH₃CN was removed under reduced pressure and the remainingsolvent was removed by lyophilization to give the product (40 mg, yield38%). ¹H NMR (MeOD 400 MHz): δ 9.42 (s, 1H), 8.13 (t, J=7.6 Hz, 1H),8.02-7.95 (m, 2H), 7.86 (d, J=11.2 Hz, 1H), 7.62 (dd, J=9.2, 2.4 Hz,1H), 7.32 (d, J=2.4 Hz, 1H). MS (ESI): m/z 284.9 [M+H]⁺.

Example 51 Compound V-7:1-(6-hydroxyquinazolin-2-yl)piperidine-4-carboxylic acid

Step 1: Synthesis of methyl1-(6-methoxyquinazolin-2-yl)piperidine-4-carboxylate

A mixture of 2-chloro-6-methoxyquinazoline (Intermediate 8, 700 mg, 3.60mmol), methyl 4-piperidinecarboxylate (2.58 g, 18.0 mmol) and Et₃N (729mg, 7.20 mmol) in ^(i)PrOH (15 mL) was heated to 50° C. for 16 hours.The reaction mixture was diluted with EtOAc (100 mL) and washed with H₂O(30 mL×2) and brine (30 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to give the crude product. The crudeproduct was purified by prep-HPLC (0.1% TFA as additive). Most of CH₃CNwas removed under reduced pressure and the remaining solvent was removedby lyophilization to give the product (1.10 g, yield: 73%).

Step 2: Synthesis of methyl1-(6-hydroxyquinazolin-2-yl)piperidine-4-carboxylate

Followed Scheme 6, Step 2, B conditions for BBr₃ deprotection.

Step 3: Synthesis of 1-(6-hydroxyquinazolin-2-yl)piperidine-4-carboxylicacid

To a solution of methyl1-(6-hydroxyquinazolin-2-yl)piperidine-4-carboxylate (330 mg, crude fromabove) in MeOH (10 mL) was added aqueous NaOH (10 mL, 2M). The mixturewas stirred at 25° C. for 16 hours. The reaction mixture was acidifiedwith 1N HCl to pH=1˜2, then concentrated to give a residue, which wasdiluted with EtOAc (100 mL), filtered and concentrated to give the crudeproduct which was purified by prep-HPLC (0.1% TFA as additive). Most ofthe solvent was removed in vacuo followed by lyophilization to give theproduct (70 mg, 2-step yield: 19%) as a solid. ¹H NMR (DMSO-d₆ 400 MHz):δ 9.15 (s, 1H), 7.51 (d, J=9.2 Hz, 1H), 7.37 (dd, J=9.2, 2.8 Hz, 1H),7.13 (d, J=2.8 Hz, 1H), 4.59 (d, J=13.2 Hz, 2H), 3.16 (t, J=11.4 Hz,2H), 2.68-2.52 (m, 1H), 1.93 (dd, J=13.2, 3.2 Hz, 2H), 1.62-1.48 (m,2H). MS (ESI): m/z 274.1 [M+H]⁺.

Example 52 Compound VI-1:3-chloro-4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)benzoic acid

Step 1: Synthesis of3-chloro-4-(6-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)benzoic acid

A mixture of 2-chloro-6-methoxy-3-(trifluoromethyl)quinoxaline(Intermediate 11, 150 mg, 0.571 mmol), 4-carboxy-2-chlorophenylboronicacid (115 mg, 0.571 mmol), K₂CO₃ (160 mg, 1.14 mmol) and Pd(dppf)Cl₂ (50mg, 0.0612 mmol) in diethylene glycol monomethyl ether (2 mL) and water(0.6 mL) was stirred at 120° C. under N₂ atmosphere for 2 hours. Theresulting mixture was cooled to room temperature and diluted with water(50 mL), acidified with aqueous HCl (2M) until pH=5, extracted withEtOAc (50 mL×3), dried over Na₂SO₄ and concentrated in vacuo. Theresidue was purified by column chromatography on silica gel(PE/EtOAc=1/1) to give the product (150 mg, yield 68%) as a solid.

Step 2: Synthesis of3-chloro-4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)benzoic acid

A mixture of3-chloro-4-(6-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)benzoic acid(150 mg, 0.392 mmol) and BBr₃ (3 mL) in anhydrous DCM (30 mL) wasstirred at 25° C. overnight. The resulting mixture was quenched withwater (30 mL), extracted with EtOAc (50 mL×3), dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by prep-HPLC (0.1% TFAas additive) to give Compound VI-1 (26 mg, yield 18%) as a solid. ¹H NMR(DMSO-d₆ 400 MHz): δ 11.22 (brs, 1H), 8.14 (d, J=9.2 Hz, 1H), 8.10 (d,J=1.6 Hz, 1H), 8.04 (dd, J=8.0, 1.6 Hz, 1H), 7.77 (d, J=8.0 Hz, 1H),7.70 (dd, J=9.2, 1.8 Hz, 1H), 7.47 (d, J=1.8 Hz, 1H). MS (ESI): m/z369.0 [M+H]⁺.

Example 53 Compound VI-2:4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)benzoic acid

Followed the procedure described for Compound VI-1, starting fromIntermediate 11 and 4-boronobenzoic acid. ¹H NMR (MeOH-d₄ 500 MHz): δ8.07 (d, J=8.5 Hz, 2H), 7.96 (d, J=9.0 Hz, 1H), 7.61 (d, J=8.5 Hz, 2H),7.51 (dd, J=2.5, 9.5 Hz; 1H), 7.35 (d, J=2.5 Hz, 1H); MS (ESI): m/z 335[M+1]⁺.

Example 54 Compound VI-3:2-(4-(2H-tetrazol-5-yl)phenyl)-3-(trifluoromethyl) quinoxalin-6-ol

Step 1: Synthesis of2-(4-(2H-tetrazol-5-yl)phenyl)-6-methoxy-3-(trifluoromethyl)quinoxaline

2-chloro-6-methoxy-3-(trifluoromethyl)quinoxaline (Intermediate 11, 321mg) was treated with (4-(2H-tetrazol-5-yl)phenyl)boronic acid (256 mg),K₂CO₃ (338 mg) and PdCl₂(dppf) (27 mg) in diethylene glycol methyl ether(3 mL) and water (0.8 mL) in a sealed microwave tube and heated at 120°C. with microwave for 1.5 h. The resultant mixture was diluted withwater (20 mL) and acidifed to pH 4 to precipitate the desired product.The solids were collected by filtration, dried under vacuum andtriturated with DCM to give 360 mg of gray solid.

Step 2: Synthesis of Compound VI-3

Above product was treated with excess BBr₃ in DCM over night. Afterremoval of all solvents, the resultant mixture was suspended in NaHCO₃solution and stirred for a couple of hours. The solids were collected,dried and triturated with DCM to afford the desired product. ¹H NMR(DMSO-d₆ 300 MHz): δ 8.16-8.21 (m, 3H), 7.80 (1H, d, J=9 Hz), 7.61 (1H,dd, J=3 and 9 hz), 7.13 (1H, d, J=9 Hz), 7.40 (1H, d, J=3 Hz). MS (ESI):m/z 359.0 [M+1]⁺.

Example 55 Compound VI-4:1-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)piperidine-4-carboxylicacid

Step 1: Synthesis of ethyl1-(6-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)piperidine-4-carboxylate

2-chloro-6-methoxy-3-(trifluoromethyl)quinoxaline (Intermediate 11, 240mg) was suspended in MeCN (1.5 mL) with ethyl piperidine-4-carboxylate(173 mg) and TEA (205 mg) in a sealed microwave tube and heated at 150°C. with microwave for three hours. The resultant mixture was purified bysilica gel column chromatography to afford 310 mg of ethyl1-(6-methoxy-3-(trifluoromethyl) quinoxalin-2-yl)piperidine-4-carboxylate.

Step 2: Synthesis of Compound VI-4

Followed BBr₃ deprotection described in Step 2 of Compound VI-3, withfurther purification by column chromatography.

¹H NMR (DMSO-d₆ 300 MHz): δ 10.52 (1H, s), 7.80 (1H, d, J=12 Hz), 7.47(1H, dd, J=3, 12 Hz), 7.26 (1H, d, J=12 Hz), 3.50 (2H, d, J=12 hz), 2.95(2H, dd, J=12 Hz), 2.44 (1H, m), 1.95 (2H, m), 1.75 (2H, m) ppm. MS(ESI): m/z 342.0.0 [M+1]⁺.

Example 56 Compound VI-5,4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)cyclohex-3-enecarboxylicacid

2-Chloro-3-(trifluoromethyl)quinoxalin-6-ol (Intermediate 14, 109 mg)was suspended with K₂CO₃ (97 mg), ethyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-enecarboxylate(117 mg), PdCl₂(dppf) (13 mg) in dioxane (1 mL) and water (0.2 mL) in asealed microwave tube and heated at 100° C. over 40 min with microwave.The resultant mixture was diluted with water (5 mL) and MeOH (2 mL) andtreated with LiOH (65 mg) overnight. After filtration through a pad ofCelite, the clear aqueous layer was acidified with 1N HCl to pH 3. Afterthe suspended solution was stirred over 1 h, filtration and rinsing withwater afforded the desired product (125 mg). ¹H NMR (DMSO-d₆ 300 MHz): δ12.30 (1H, s), 10.93 (1H, s), 8.04 (1H, d, J=9 Hz), 7.59 (1H, dd, J=3, 9hz), 7.35 (1H, d, J=3 Hz), 5.84 (1H, s), 2.58 (1H, m), 2.34 (4H, m),2.07 (1H, m), 1.75 (1H, m) ppm. MS (ESI): m/z 339.0 [M+1]⁺.

Example 57 Compound VI-6: 4-(6-hydroxyquinoxalin-2-yl)benzoic acid

Step 1: Synthesis of methyl 4-(6-methoxyquinoxalin-2-yl)benzoate

To a solution of methyl 4-acetylbenzoate (1.00 g, 5.61 mmol) in DMSO (5mL) was slowly added 40% HBr (1.5 mL) at 20° C. under stirring. Themixture was stirred at 60° C. for 12 hours. The mixture was diluted withwater (10 mL) and the resulting mixture was extracted with DCM (10mL×3), washed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated in vacuo to give methyl 4-(2-oxoacetyl)benzoate (150 mg),which was taken forward without further purification. To a solution of4-methoxybenzene-1,2-diamine (107 mg, 0.78 mmol) in 5 mL of acetic acidwas added the crude methyl 4-(2-oxoacetyl)benzoate (150 mg). The mixturewas refluxed for 2.5 hours. The solvent was removed in vacuo. The crudeproduct was purified by prep-TLC (PE/EA: 1/1) to separate the desiredmethyl 4-(6-methoxyquinoxalin-2-yl)benzoate (more polar) from theundesired methyl 4-(7-methoxyquinoxalin-2-yl)benzoate. The twostructures were confirmed by HMBC spectra.

Step 2: Synthesis of 4-(6-methoxyquinoxalin-2-yl)benzoic acid

A mixture of methyl 4-(6-methoxyquinoxalin-2-yl)benzoate (80 mg, 0.272mmol) and aqueous NaOH (5 mL, 2M) in MeOH (5 mL) was stirred at 30° C.overnight. The resulting mixture was concentrated in vacuo. The residuewas acidified with aqueous HCl (2M) until pH=3 and concentrated. Thesolid was dissolved in MeOH (30 mL) and filtered. The filtrate wasconcentrated in vacuo to give crude product (80 mg), which was used fornext step directly without further purification.

Step 3: Synthesis of 4-(6-hydroxyquinoxalin-2-yl)benzoic acid

Followed the procedure described in step 2 of Compound VI-1. ¹H NMR(DMSO-d₆ 400 MHz): δ 13.16 (brs, 1H), 10.68 (brs, 1H), 9.50 (s, 1H),8.41 (d, J=8.4 Hz, 2H), 8.12 (d, J=8.4 Hz, 2H), 8.03 (d, J=8.8 Hz, 1H),7.47 (dd, J=8.8, 2.8 Hz, 1H), 7.32 (d, J=2.8 Hz, 1H). MS (ESI): m/z266.7 [M+H]⁺.

Example 58 Compound VII-1:3-(4-carboxyphenyl)-7-hydroxybenzo[e][1,2,4]triazine 1-oxide

Step 1: Synthesis of 3-amino-7-hydroxybenzo[e][1,2,4]triazine 1-oxide

This intermediate was prepared according to the published method in WO2006024034 starting from 4-amino-3-nitrophenol.

Step 2: Synthesis of 3,7-dihydroxybenzo[e][1,2,4]triazine 1-oxide

Sodium nitrite (22 mmol) was added in small portions to a stirredsolution of 3-amino-7-hydroxybenzo[e][1,2,4]triazine 1-oxide (20 mmol)in TFA (40 mL) at 5° C., and the solution stirred at 20° C. for 3 h. Thesolution was poured into ice/water (400 mL), stirred 30 min, filtered,washed with water (3×10 mL) and dried.

Step 3: Synthesis of 3-chloro-7-hydroxybenzo[e][1,2,4]triazine 1-oxide

1 g of 3,7-dihydroxybenzo[e][1,2,4]triazine 1-oxide was suspended inPOCl₃ (20 mL) and DMF (0.1 mL) and stirred at 100° C. for 2 h. Thesolution was cooled, poured into ice/water (200 mL), stirred for 30 min,filtered, washed with water (3×10 mL) and dried.

Step 4: Synthesis of3-(4-carboxyphenyl)-7-hydroxybenzo[e][1,2,4]triazine 1-oxide

3-chloro-7-hydroxybenzo[e][1,2,4]triazine 1-oxide (2 mmol) was mixedwith 1.0 equivalent of 4-(methoxycarbonyl)phenylboronic acid and 2equivalents of K₂CO₃ in 20 ml of 50% dioxane/water. A catalytic amountof Pd(PPh₃)₄ was added and the mixture was heated to reflux for 2 hours.The reaction was concentrated and re-dissolved in 20 ml water and 5 ml1N NaOH was added to hydrolyze the ester. The mixture was extracted withDCM (3×10 ml). The aqueous layer was filtered and the filtrate wasneutralized with 1N HCl to pH=5. The precipitate was filtered and washedwith water and dried. 500 mg of product was obtained. ¹H NMR (DMSO-d6400 MHz): δ 11.38 (s, 1H), 8.44 (d, 2H), 8.15 (m, 3H), 7.67 (d, 1H),7.60 (s, 1H). MS (ESI): m/z 284.5 [M+1]⁺.

Example 59 Compound VII-2:4-(7-hydroxybenzo[e][1,2,4]triazin-3-yl)benzoic acid

3-(4-Carboxyphenyl)-7-hydroxybenzo[e][1,2,4]triazine 1-oxide (CompoundVII-1, 350 mg, 1.23 mmol) was dissolved in 70% ethanol in water (20 ml)at reflux. 5.0 equivalents of Na₂S₂O₄ were added. The reaction turneddark brown. After 30 minutes, the solution was concentrated to removeethanol and the solid was filtered and washed with water and dried (12mg). ¹H NMR (DMSO-d6 400 MHz): δ 8.67 (d, 2H), 8.16 (d, 2H), 8.09 (d,1H), 7.77 (d, 1H), 7.67 (s, 1H). MS (ESI): m/z 268.43 [M+1]⁺.

Example 60 Compound VII-3:3-(4-carboxy-2-fluorophenyl)-7-hydroxybenzo[e][1,2,4]triazine 1-oxide

Prepared following the procedure described for Compound VII-1 where theboronic acid used in Step 4 was2-fluoro-4-(methoxycarbonyl)phenylboronic acid and using PdCl₂(dppf) asthe catalyst. ¹H NMR (DMSO-d6 500 MHz TMS): δ 8.12 (m, 2H), 7.92 (dd,1H), 7.77 (d, 1H) 7.67 (dd, 1H), 7.61 (d, 1H); MS (ESI): m/z 302.52[M+1]⁺.

Example 61 Compound VII-4:3-fluoro-4-(7-hydroxybenzo[e][1,2,4]triazin-3-yl)benzoic acid

Prepared following the procedure described for Compound VII-2, startingfrom 3-(4-carboxy-2-fluorophenyl)-7-hydroxybenzo[e][1,2,4]triazine1-oxide (Compound VII-3). ¹H NMR (DMSO-d₆ 500 MHz): δ 8.22 (t, 1H), 8.10(d, 1H), 7.94 (dd, 1H), 7.82 (dd, 2H), 7.70 (d, 1H). MS (ESI): m/z286.52 [M+1]⁺.

Example 62 Compound VIII-1: 4-(7-hydroxyisoquinolin-3-yl)benzoic acid

Step 1: Synthesis of 4-(7-methoxyisoquinolin-3-yl)benzoic acid

Followed the procedure described in Compound W-8 step 1, starting with3-chloro-7-methoxyisoquinoline (Intermediate 12) and 4-boronobenzoicacid with an alternative isolation procedure: After reaction wascomplete, the reaction mixture was diluted with H₂O (10 mL). The aqueouslayer was extracted with EtOAc/MeOH (v/v=10:1, 30 mL×2). Then theaqueous layer was acidified with 2M HCl to pH=6. The precipitate wascollected by filtration and washed with MeOH (10 mL), dried under highvacuum to give the product.

Step 2: Synthesis of Compound VIII-1

Followed the procedure described in Compound W-8, step 2. ¹H NMR(DMSO-d₆ 400 MHz): δ 9.27 (s, 1H), 8.45 (s, 1H), 8.25 (d, J=8.4 Hz, 2H),8.05 (d, J=8.4 Hz, 2H), 7.95 (d, J=8.8 Hz, 1H), 7.42 (dd, J=8.8, 2.4 Hz,1H), 7.37 (d, J=2.0 Hz, 1H). MS (ESI): m/z 265.9 [M+H]⁺.

Example 63 Compound IX-1: 4-(7-hydroxyquinolin-3-yl)benzoic acid

Step 1: Synthesis of 4-(7-methoxyquinolin-3-yl)benzoic acid

To a mixture of 3-bromo-7-methoxyquinoline (Intermediate 13, 380 mg, 1.6mmol), 4-boronobenzoic acid (266 mg, 1.6 mmol) and Na₂CO₃ (848 mg, 8.0mmol) in DME/H₂O/EtOH (5 mL, V/V/V=1/1/0.5) was added Pd(dppf)Cl₂ (585mg, 0.8 mmol). The mixture was heated to 120° C. by microwave for 1 h.The mixture was partitioned between water (20 mL) and ethyl acetate (20mL). The aqueous phase was separated and acidified to pH=3 with 1 N HCl.The precipitate was filtered and dried in vacuo to afford product as apowder (130 mg, 29.1%).

Step 2: Synthesis of Compound IX-1

To a solution of above product (130 mg, 0.46 mmol) in DCM (3 mL) wasadded carefully BBr₃ (0.4 mL) at room temperature. The mixture wasstirred overnight. Water (5 mL) was added carefully. The volatiles wereevaporated and the residue was purified by prep-HPLC to afford CompoundIX-1 as a powder (80 mg, 65.6%). ¹HNMR (MeOH-d₄ 500 MHz): δ 9.35 (s,1H), 9.25 (s, 1H), 8.24 (d, J=8.5 Hz, 3H), 8.00 (d, J=8.0 Hz, 2H), 7.53(d, J=1.5 Hz, 1H), 7.45 (s, 1H); MS (ESI): m/z 266.0 [M+1]⁺.

Example 64 Compound IX-2: 3-(4-carboxyphenyl)-7-hydroxyquinoline 1-oxide

Step 1: Synthesis of methyl 4-(7-methoxyquinolin-3-yl)benzoate

Followed Step 1 of Compound IV-1, starting from4-(methoxycarbonyl)phenylboronic acid and Intermediate 13, and where thebase used was Na₂CO₃, the solvent DME/H₂O (8:2), and the reaction wasrun at 100° C. for 2 h.

Step 2: Synthesis of 7-methoxy-3-(4-(methoxycarbonyl)phenyl)quinoline1-oxide

A mixture of methyl 4-(7-methoxyquinolin-3-yl)benzoate (632 mg, 2.16mmol), m-CPBA (742 mg, 4.31 mmol) and DCM (10 mL) was stirred overnight.Saturated Na₂CO₃ solution was added and adjusted the pH>7, the organiclayer was washed with water (50 mL×2), dried over NaSO₄ and evaporatedin vacuo to give the crude product as a yellow solid (540 mg, 81.0%).

Step 3: Synthesis of Compound IX-2

To a solution of the above product (310 mg, 1.0 mmol) in DCM (15 mL) wasadded BBr₃ (1.0 mL, 10.6 mmol) at 0° C. The reaction mixture was warmedto room temp. and stirred for 48 h. The mixture was poured intoice-water, filtered and washed with water, dried and purified by prepHPLC to give Compound IX-2 (25.0 mg, 8.87%). H-NMR (DMSO-d₆ 500 MHz):8.94 (s, 1H), 8.26 (s, 1H), 8.01˜8.06 (m, 3H), 7.92˜7.94 (d, J=8.0 Hz,2H), 7.81 (s, 1H). MS (ESI): m/z=282.0 [M+1]⁺.

Example 65 Compound VIII-2:3-fluoro-4-(7-hydroxyisoquinolin-3-yl)benzoic acid

Step 1: A mixture of 3-chloro-7-methoxyisoquinoline (Intermediate 12)(300 mg, 1.55 mmol), 4-carboxy-2-fluorophenylboronic acid (285 mg, 1.55mmol), K₂CO₃ (642 mg, 4.65 mmol) and Pd(dppf)Cl₂ (30 mg, 0.037 mmol) inDEGME (7 mL) and water (2 mL) was stirred under N₂ atmosphere at 120° C.for 3 hours. The resulting mixture was cooled to room temperature andfiltered. The filtrate was acidified with aqueous HCl (2M) until pH=6and filtered. The solid was dried under reduced pressure to give3-fluoro-4-(7-methoxyisoquinolin-3-yl)benzoic acid (140 mg, yield: 30%)as off-white solid.

Step 2: A mixture of the above product (70 mg, 0.24 mmol) and BBr₃ (0.2mL, 2.1 mmol) in anhydrous DCM (2 mL) was stirred at 15° C. overnight.The resulting mixture was quenched with water (10 mL) and basified withaqueous sat. NaHCO₃ until pH=8. The mixture was concentrated in vacuoand purified by prep-HPLC (0.1% TFA as additive) to give Compound VIII-2(12 mg, yield 18%) as yellow solid. ¹H NMR (CD₃OD 400 MHz TMS): δ 9.41(s, 1H), 8.43 (s, 1H), 8.10 (d, J=8.8 Hz, 1H), 8.08-7.96 (m, 2H), 7.93(dd, J=11.6, 0.8 Hz, 1H), 7.65 (dd, J=8.8, 2.4 Hz, 1H), 7.56 (d, J=2.4Hz, 1H). MS (ESI): m/z 283.7 [M+H]⁺.

Example 66 Compound VIII-3:4-(7-hydroxy-4-methylisoquinolin-3-yl)benzoic acid

Step 1: To a mixture of 3-chloro-7-methoxy-4-methylisoquinoline(Intermediate 15) (470 mg, 2.27 mmol), 4-carboxyphenylboronic acid (451mg, 2.72 mmol) and K₂CO₃ (627 mg, 4.54 mmol) in DEGME/H₂O (7 mL/1 mL)was added Pd(dppf)Cl₂ (40.8 mg, 0.050 mmol) under N₂ atmosphere. Thenthe mixture was heated to 120° C. for 16 hours. H₂O (30 mL) was added,and the resulting mixture was extracted with EtOAc (15 mL×2). Theaqueous layer was neutralized with 1N aqueous HCl to pH=7, extractedwith EtOAc (20 mL×3). The combined organic layers were washed with H₂O(20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated. Purification by silica gel column (PE/EtOAc=20/1 to 1/1,then EtOAc) gave 4-(7-methoxy-4-methylisoquinolin-3-yl)benzoic acid(85.0 mg, yield 13%).

Step 2: To a solution of the above product (85.0 mg, 0.290 mmol) inanhydrous DCM (2 mL) was added BBr₃ (0.20 mL, 2.1 mmol, d=2.64 g/mL)dropwise at 0° C., then stirred at 25° C. for 16 hours. The reactionmixture was quenched with H₂O (20 mL) and neutralized with aqueous NaOH(2M) to pH=6-7, followed by DCM extraction/workup. The crude product wastriturated with EtOAc (15 mL) to give Compound II-25 (20 mg, yield 25%)as an off-white solid. ¹H NMR (MeOD₄ 400 MHz): δ 8.94 (s, 1H), 8.15 (d,J=8.0 Hz, 2H), 8.08 (d, J=8.8 Hz, 1H), 7.63 (d, J=8.0 Hz, 2H), 7.46 (dd,J=9.2, 2.4 Hz, 1H), 7.32 (d, J=2.4 Hz, 1H), 2.59 (s, 3H). MS (ESI): m/z279.6 [M+H]⁺.

Example 67 Compound VI-7:3-fluoro-4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)benzoic acid

Step 1: Followed the coupling procedure described in Step 1 of CompoundVIII-2 (Example 65), where the starting materials were2-chloro-6-methoxy-3-(trifluoromethyl)quinoxaline (Intermediate 11) and4-carboxy-2-fluorobezeneboronic acid. After H₂O quench, the aqueouslayer was acidified with 0.1M HCl to pH=1, followed by EtOAc/aqueousworkup and column purification to give3-fluoro-4-(6-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)benzoic acid(50 mg, yield 19%).

Step 2: Followed the BBr₃ deprotection method described in CompoundVIII-2 (Example 65, step 2). Compound VI-7 (16 mg, yield 33%) wasisolated as an off-white solid. ¹H NMR (MeOD 400 MHz): δ 8.09 (d, J=9.2Hz, 1H), 8.07-7.99 (m, 1H), 7.88 (d, J=8.4 Hz, 1H), 7.70-7.62 (m, 2H),7.49 (d, J=2.8 Hz, 1H). LC-MS purity: 100%. MS (ESI): m/z 352.9 [M+H]⁺.

Example 68 Compound VI-8:(1r,4r)-4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)cyclohexanecarboxylicacid

Step 1: Followed the coupling procedure similar to that described inCompound VIII-2 (Example 65), where the starting materials were ethyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-enecarboxylateand 2-chloro-6-methoxy-3-(trifluoromethyl)quinoxaline (Intermediate 11)and where the reaction here was stirred under N₂ atmosphere at 100° C.for 3 hours. Ethyl4-(6-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)cyclohex-3-enecarboxylatewas isolated (800 mg, yield 55%) as yellow solid.

Step 2: A mixture of the above product (200 mg, 0.526 mmol) and 10% Pd/C(100 mg, 50% wet) in EtOH (10 mL) was stirred under H₂ (50 psi) at 30°C. for 2 days. The mixture was filtered and the filtrate wasconcentrated to give crude ethyl4-(6-methoxy-3-(trifluoromethyl)-1,2,3,4-tetrahydroquinoxalin-2-yl)cyclohexanecarboxylate(190 mg).

Step 3: A mixture of the above product (crude 190 mg) and MnO₂ (200 mg,2.30 mmol) in DCM (20 mL) was stirred at 10° C. overnight. The mixturewas filtered and the filtrate was concentrated followed by purificationby silica gel column (PE/EtOAc=40/1) to give ethyl4-(6-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)cyclohexanecarboxylate(180 mg, 2-step yield: 90%) as colorless oil.

Step 4: Followed the BBr₃ deprotection method described in CompoundVIII-3 (Example 66, step 2) to give Compound VI-8 (trans) (23 mg, yield12%) as an off-white solid and Compound VI-9 (cis) (22 mg, yield 12%) asan off-white solid after prep-HPLC.

¹H NMR of Compound VI-8 (DMSO-d₆ 400 MHz): δ 7.97 (d, J=9.2 Hz, 1H),7.88-7.78 (m, 1H), 7.12 (s, 1H), 3.10-2.98 (m, 1H), 2.48-2.38 (m, 2H),2.40-2.20 (m, 1H), 2.10-1.90 (m, 2H), 1.53-1.51 (m, 2H), 1.51-1.46 (m,2H). MS (ESI): m/z 340.8 [M+H]⁺.

Example 69 Compound VI-9:(1s,4s)-4-(6-hydroxy-3-(trifluoromethyl)quinoxalin-2-yl)cyclohexanecarboxylicacid

See Example 68 for synthesis. ¹H NMR (DMSO-d₆ 400 MHz): δ 12.12 (brs,1H), 10.84 (brs, 1H), 7.99 (d, J=9.2 Hz, 1H), 7.55 (dd, J=9.2, 2.4 Hz,1H), 7.31 (d, J=2.4 Hz, 1H), 3.08-2.94 (m, 1H), 2.36-2.28 (m, 1H),2.10-2.00 (m, 2H), 1.92-1.70 (m, 4H), 1.54-1.38 (m, 2H). MS (ESI): m/z340.7 [M+H]⁺.

Example 70 Compound IV-45:3-chloro-4-(5-fluoro-6-hydroxyquinolin-2-yl)benzoic acid

2-Chloro-5-fluoroquinolin-6-ol (Intermediate 16, 1.0 mmol) and4-borono-3-chlorobenzoic acid (1.1 mmol) were mixed with 5% (Ph₃P)₄Pdand 4.0 mmol of NaHCO₃ in 20 ml 50% dioxane/water. The mixture wasdegassed three times by evacuation and argon filling and heated at 95°C. overnight. The reaction was diluted with 20 ml of water and filtered.The filtrate was acidified with 1N HCl to pH=4. The precipitate wasfiltered and washed with water, dried. The crude was purified by columnusing AcOH/MeOH/EtOAC (1/5/94) as solvent B and hexanes as solvent Awith gradient 2 to 100%. The fractions with compound were pooled andevaporated to obtain white solid. The solid was triturated withdiisopropyl ether. 50 mg of final product was obtained. ¹H-NMR (DMSO-d₆,300 MHz, TMS): δ 10.55 (b, 1H), 8.47 (d, 1H), 8.01-8.03 (m, 2H), 7.79(m, 3H), 7.56 (t, 1H). MS (ESI): m/z=316.3 [M−1]⁻.

Example 71 Compound IV-46:4-(5-fluoro-6-hydroxyquinolin-2-yl)-3-methylbenzoic acid

Followed the procedure described for Example 70, starting from2-chloro-5-fluoroquinolin-6-ol (Intermediate 16) and4-borono-3-methylbenzoic acid. ¹H-NMR (DMSO-d₆, 300 MHz, TMS): δ 13.03(b, 1H), 10.47 (b, 1H), 8.43 (d, 1H), 7.93 (s, 1H), 7.90 (d, 1H), 7.78(d, 1H), 7.72 (d, 1H), 7.62 (d, 1H), 7.52 (t, 1H), 2.43 (s, 3H). MS(ESI): m/z=296.38 [M−1]⁻.

Example 72 Synthesis of Intermediates Intermediate 1 Synthesis of2-chloro-6-methoxyquinoline Step 1: Synthesis of 6-methoxyquinoline1-oxide

To a solution of 6-methoxyquinoline (2.00 g, 12.6 mmol) in AcOH (10 mL)was added H₂O₂ (30% in water, 1.9 mL, 18.9 mmol), the mixture was heatedto 70° C. for 21 hours. The mixture was basified with 2M NaOH to pH 8-9and extracted with CH₂Cl₂ (200 mL), the combined organic layer waswashed with brine (50 mL), dried over Na₂SO₄, filtered and concentratedto give the crude product, which was purified by silica gel column(EtOAc/MeOH=10/1) to give 6-methoxyquinoline 1-oxide (1.20 g, 55%) as asolid.

Step 2: Synthesis of 6-methoxyquinolin-2-ol

A solution of 6-methoxyquinoline 1-oxide (300 mg, 1.71 mmol) in Ac₂O(5.0 mL) was refluxed for 2 hours. The solvent was removed under reducedpressure. The residue was dissolved in EtOAc (50 mL), the organic layerwas washed with brine (10 mL), dried over Na₂SO₄, filtered andconcentrated to give the crude product which was purified by silica gelcolumn (PE/EtOAc=1/2) to give 6-methoxyquinolin-2-ol (200 mg, yield67%).

Step 3: Synthesis of 2-chloro-6-methoxyquinoline

A solution of 6-methoxyquinolin-2-ol (400 mg, 2.29 mmol) in POCl₃ (5.0mL) was refluxed for 2 hours. The solvent was removed under reducedpressure and the residue was dissolved in EtOAc (50 mL), the organiclayer was washed with saturated NaHCO₃ (30 mL×2), dried over Na₂SO₄,filtered and concentrated to give the crude product, which was purifiedby silica gel column (PE/EtOAc=10/1) to give 2-chloro-6-methoxyquinoline(380 mg, yield 86%) as a solid. ¹H NMR (CDCl3 400 MHz): δ 7.92 (d, J=8.4Hz, 1H), 7.85 (d, J=9.2 Hz, 1H), 7.32-7.25 (m, 2H), 7.00 (d, J=2.4 Hz,1H), 3.86 (s, 3H).

Intermediate 2: Synthesis of 2-chloroquinolin-6-ol

To a solution of 2-chloro-6-methoxyquinoline (Intermediate 1) (2.00 g,10.4 mmol) in anhydrous DCM (100 mL) was added BBr₃ (6 mL, 62.2 mmol)dropwise at 0° C. The reaction mixture was stirred at 25° C. for 2hours, then quenched with aqueous saturated NH₄Cl (50 mL) and filtered.The filtrate was extracted with CH₂Cl₂/MeOH (v/v=10/1, 30 mL×2) and thecombined organic layers were washed with brine (30 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give2-chloroquinolin-6-ol (1.30 g, yield 70%) as yellow solid. ¹H NMR (CDCl₃300 MHz): δ 7.95 (t, J=8.1 Hz, 2H), 7.35 (dd, J=6.0, 3.3 Hz, 2H), 7.13(d, J=2.7 Hz, 1H).

Intermediate 3: Synthesis of 2,4-dichloro-3-fluoro-6-methoxyquinolineStep 1: Synthesis of 2-fluoromalonic acid

To a solution of diethyl 2-fluoromalonate (5.00 g, 28.1 mmol) in EtOH(100 mL) was added LiOH.H₂O (2.70 g, 64.3 mmol) at 25° C. The mixturewas heated to 50° C. for 16 hours. The mixture was filtered to collectsolid. The filtrate was concentrated to dryness to get oil. The oil andthe solid were dissolved in H₂O (30 mL) and MTBE (100 mL), the mixturewas acidified by adding conc. HCl to pH 1, the aqueous layer wasextracted with MTBE (30 mL×2), the combined organic layers were driedover Na₂SO₄, filtered and concentrated to give 2-fluoromalonic acid(3.00 g, yield 88%) as a solid.

Step 2: Synthesis of 2,4-dichloro-3-fluoro-6-methoxyquinoline

A suspension of fluoromalonic acid (1.00 g, 8.13 mmol) in POCl₃ (10 mL)was heated to 85° C. to dissolve the solid. The mixture was cooled to60° C. and p-anisidine (900 mg, 7.32 mmol) was added portion wise over 1hour. After addition, the reaction mixture was refluxed for 2 hours. Thesolvent was removed in vacuo. The mixture was diluted with ice water andbasified by adding NH₃—H₂O to pH 9. The aqueous layer was extracted withEtOAc (30 mL×3), the combined organic layers were washed with brine (20mL), dried over Na₂SO₄, filtered and concentrated to give the crudeproduct, which was purified by silica gel column (PE/EtOAc=10/1) to give2,4-dichloro-3-fluoro-6-methoxyquinoline (650 mg, yield 36%).

¹H NMR (CDCl₃ 300 MHz): δ 7.92 (d, J=9.0 Hz, 1H), 7.41-7.31 (m, 2H),4.00 (s, 3H).

Intermediate 4 Synthesis of 2,3-dichloro-6-methoxyquinoline Step 1:Synthesis of 2-chloro-N-(4-methoxyphenyl)acetamide

A mixture of 4-methoxyaniline (5.00 g, 40.6 mmol), chloroacetic acid(8.6 g, 91.5 mmol), EDCI (12.0 g, 61.2 mmol), HOBT (8.4 g, 61.3 mmol)and NMM (13 mL, 122 mmol) in anhydrous CH₂Cl₂ (50 mL) was stirred at 30°C. for 3 hours. The mixture was quenched with ice water, and thenextracted with CH₂Cl₂ (30 mL×2). The combined organic layers were washedwith brine (30 mL), dried over Na₂SO₄, filtered and concentrated to givethe crude product, which was purified by silica gel column(PE/EtOAc=5/1) to give the product (1.60 g, yield 20%).

Step 2: Synthesis of 2,3-dichloro-6-methoxyquinoline

POCl₃ (1.6 mL, 17.5 mmol) was added dropwise to DMF (0.29 mL, 3.80 mmol)at 0° C. After addition, 2-chloro-N-(4-methoxyphenyl)acetamide (500 mg,2.50 mmol) was added portionwise. The mixture was stirred at 25° C. for15 minutes and heated to 75° C. for 3 hours. The reaction mixture wasquenched with ice water and neutralized by 2M NaOH to pH 7. An aqueousworkup with EtOAc was followed by purification by silica gel column(PE/EtOAc=20/1) to give Intermediate 4 (50 mg, yield 9%). ¹H NMR (CDCl₃400 MHz): δ 8.06 (s, 1H), 7.81 (d, J=9.6 Hz, 1H), 7.30 (dd, J=9.2, 2.8Hz, 1H), 6.92 (d, J=2.8 Hz, 1H), 3.87 (s, 3H).

Intermediate 5 Synthesis of methyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)benzoate

To a mixture of methyl 4-bromo-3-(trifluoromethyl)benzoate (1.00 g, 3.53mmol), bis(pinacolato)diboron (1.79 g, 7.06 mmol), and KOAc (1.04 g,10.6 mmol) in DMSO (15 mL) was added Pd(PPh₃)₄ (816 mg, 0.706 mmol)under N₂ atmosphere. Then the mixture was heated to 120° C. for 3 hours.The reaction mixture was cooled followed by an aqueous/EtOAc workup togive the crude product (2.3 g) as a yellow oil.

Intermediate 6 Synthesis of 2-chloro-3-(trifluoromethyl)quinolin-6-olStep 1: Synthesis of (5-methoxy-2-nitrophenyl)methanol

To a solution of 5-methoxy-2-nitrobenzoic acid (20.0 g, 0.102 mol) inanhydrous THF (200 mL) was added SOCl₂ (20 mL), the mixture was refluxedfor 4 hours. The solvent was removed under reduced pressure and theresidue was dissolved in anhydrous THF (100 mL). The solution was addeddropwise to a suspension of NaBH₄ (7.70 g, 0.202 mol) in anhydrous THF(100 mL) and DMF (140 mL) at 0° C. over a period of 30 minutes. Themixture was stirred at 30° C. for 3 hours, then quenched with ice-water(100 mL) and basified by 2M NaOH to pH 8. An EtOAc workup was followedby removal of solvent in vacuo and purification by column chromatography(PE/EtOAc=1/1) to give the product (12.0 g, yield 66%).

Step 2: Synthesis oftert-butyl(5-methoxy-2-nitrobenzyloxy)dimethylsilane

To a solution of (5-methoxy-2-nitrophenyl)methanol (12.0 g, 65.6 mmol)in anhydrous THF (200 mL) and DMF (20 mL) was added imidazole (9.80 g,144 mmol). Then TBSCl (11.8 g, 78.6 mmol) was added portionwise at 0° C.and the mixture was stirred at 30° C. for 2 hours. The mixture wasquenched with ice-water (100 mL). An EtOAc workup was followed byremoval of solvent in vacuo and purification by column chromatography(PE/EtOAc=10/1) to give the product (16.0 g, yield 84%) as a yellow oil.

Step 3: Synthesis of2-((tert-butyldimethylsilyloxy)methyl)-4-methoxyaniline

To a solution of tert-butyl(5-methoxy-2-nitrobenzyloxy)dimethylsilane(14.0 g, 47.1 mmol) in EtOH (200 mL) was added 10% Pd/C (1.40 g), themixture was stirred at 30° C. for 2 hours under H₂ (40 psi). The solidswere filtered off and the filtrate was concentrated under reducedpressure to give the product (14.0 g) as a yellow oil. ¹H NMR (DMSO 400MHz): δ 6.75 (d, J=2.4 Hz, 1H), 6.60-6.55 (m, 2H), 4.55 (s, 2H), 4.40(brs, 2H), 3.61 (s, 3H), 0.90 (s, 9H), 0.09 (s, 6H).

Step 4: Synthesis ofN-(2-((tert-butyldimethylsilyloxy)methyl)-4-methoxyphenyl)-3,3,3-trifluoropropanamide

A mixture of 2-((tert-butyldimethylsilyloxy)methyl)-4-methoxyaniline(14.0 g), 3,3,3-trifluoro-propionic acid (7.30 g, 57.0 mmol), EDCI (15.0g, 76.5 mmol), HOBT (11.0 g, 80.2 mmol) and NMM (22 mL, 157 mmol) inanhydrous CH₂Cl₂ (200 mL) was stirred at 20° C. for 2 hours. The mixturewas diluted with CH₂Cl₂ (100 mL), the organic layer was washed with 1MHCl (100 mL), H₂O (100 mL) and brine (100 mL), dried over Na₂SO₄ andconcentrated to give the product.

Step 5: Synthesis of3,3,3-trifluoro-N-(2-(hydroxymethyl)-4-methoxyphenyl)propanamide

To a solution ofN-(2-((tert-butyldimethylsilyloxy)methyl)-4-methoxyphenyl)-3,3,3-trifluoropropanamide(20.0 g) in anhydrous THF (200 mL) was added a solution of TBAF (16.6 g,63.6 mmol) in anhydrous THF (60 mL). The mixture was stirred at 35° C.for 30 minutes. The mixture was quenched with ice-water. Anaqueous/EtOAc workup was followed by removal of volatiles in vacuo. Thecrude product was purified by column chromatography on silica gel(PE/EtOAc=1/2) to give the product.

¹H NMR (CDCl3 400 MHz): δ 8.60 (brs, 1H), 7.82 (d, J=8.8 Hz, 1H), 6.85(dd, J=9.2, 3.2 Hz, 1H), 6.75 (d, J=2.8 Hz, 1H), 4.65 (s, 2H), 3.80 (s,3H), 3.23 (q, J=10.4 Hz, 2H).

Step 6: Synthesis of3,3,3-trifluoro-N-(2-formyl-4-methoxyphenyl)propanamide

To a solution of3,3,3-trifluoro-N-(2-(hydroxymethyl)-4-methoxyphenyl)propanamide (6.30g, 23.9 mmol) in anhydrous CH₂Cl₂ (200 mL) was added MnO₂ (20.0 g, 229mmol). The mixture was refluxed for 16 hours. The mixture was filteredoff and the filtrate was concentrated to give the product (5.30 g, yield84%).

Step 7: Synthesis of 6-methoxy-3-(trifluoromethyl)quinolin-2(1H)-one

To a solution of 3,3,3-trifluoro-N-(2-formyl-4-methoxyphenyl)propanamide(5.30 g, 20.3 mmol) in DMF (100 mL) was added K₂CO₃ (14.0 g, 101 mmol).The mixture was heated to 60° C. for 1.5 hours. The mixture was dilutedwith EtOAc (200 mL) and filtered off. The filtrate was washed with H₂O(100 mL) and brine (100 mL), dried over Na₂SO₄, and concentrated invacuo to give the product (4.60 g, yield 94%). ¹H NMR (CDCl3 300 MHz): δ12.25 (brs, 1H), 8.18 (s, 1H), 7.40 (d, J=9.0 Hz, 1H), 7.25 (dd, J=9.0,2.7 Hz, 1H), 7.02 (d, J=2.7 Hz, 1H), 3.87 (s, 3H).

Step 8: Synthesis of 2-chloro-6-methoxy-3-(trifluoromethyl)quinoline

A solution of 6-methoxy-3-(trifluoromethyl)quinolin-2(1H)-one (4.60 g,18.9 mmol) in POCl₃ (30 mL) was refluxed for 2.5 hours. The solvent wasremoved under reduced pressure and the residue was neutralized by 2MNaOH to pH 7. An EtOAc workup was followed by removal of volatiles underreduced pressure. The residue was purified by column chromatography onsilica gel (PE/EtOAc=15/1) to give the product (4.60 g, yield 94%).

Step 9: Synthesis of 2-chloro-3-(trifluoromethyl)quinolin-6-ol

To a solution of above product (1.00 g, 3.83 mmol) in anhydrous CH₂Cl₂(20 mL) was added BBr₃ (3.0 mL, 31.8 mmol) at 0° C. The mixture wasstirred at 0° C. for 2 hours. The mixture was quenched with ice-water(10 mL) and extracted with CH₂Cl₂ (30 mL×3). The combined organic layerwas washed with brine (50 mL), dried over Na₂SO₄ and concentrated underreduced pressure to give Intermediate 6 (600 mg, yield 63%) as a solid.¹H NMR (CDCl₃ 400 MHz): δ 8.37 (s, 1H), 8.00 (d, J=9.2 Hz, 1H), 7.48(dd, J=8.8, 2.4 Hz, 1H), 7.22 (d, J=2.8 Hz, 1H).

Intermediate 7 Synthesis of 2-chloro-4-fluoro-6-methoxyquinoline Step 1:Synthesis of 2,4-dichloro-6-methoxyquinoline

A mixture of p-anisidine (100 g, 0.813 mol), and malonic acid (85.0 g,0.817 mol) in POCl₃ (500 mL) was refluxed for 6 hours. The excess POCl₃was removed in vacuo and the residue was neutralized with 8 M NaOH to pH7. The aqueous layer was extracted with CH₂Cl₂ (300 mL×3), washed withbrine (500 mL), dried over Na₂SO₄ and concentrated under reducedpressure. Purification by column chromatography on silica gel(PE/EtOAc=15/1) gave desired (35.0 g, yield: 19%).

Step 2: Synthesis of 2-chloro-6-methoxyquinolin-4-amine

A suspension of 2,4-dichloro-6-methoxyquinoline (5.00 g, 22.0 mmol) inNH₃ (g)/MeOH (saturated, 40 mL) was heated to 150° C. for 16 hours in asealed tube. The solvent was removed and the residue was diluted withMeOH (20 mL). The mixture was filtered off and the filtrate wasconcentrated to give the crude product. Purification by columnchromatography on silica gel (PE/EtOAc=2/1) gave product (7.50 g, yield:55%) as a solid.

Step 3: Synthesis of 2-chloro-4-fluoro-6-methoxyquinoline

A solution of 2-chloro-6-methoxyquinolin-4-amine (4.50 g, 21.6 mmol) inHF-pyridine (45 mL) was cooled to −10-0° C. Then NaNO₂ (1.80 g, 26.1mmol) was added portionwise and the mixture was stirred at 0° C. for 1hour and at 30° C. for 1 hour. Then the mixture was heated to 65° C. for1.5 hours. The mixture was quenched with ice-water (100 mL); the aqueouslayer was neutralized with 2 M NaOH to pH 7. The mixture was filteredoff and the filtrate was extracted with EtOAc (50 mL×3), the organiclayer was washed with brine (100 mL), dried over Na₂SO₄ and concentratedin vacuo. Purification by column chromatography on silica gel(PE/EtOAc=15/1) gave Intermediate 7 (3.60 g, yield: 40%). ¹H NMR (CDCl3400 MHz): δ 7.92 (dd, J=9.2, 1.6 Hz, 1H), 7.40 (dd, J=9.2, 2.8 Hz, 1H),7.25 (s, 1H), 7.10 (d, J=9.2 Hz, 1H), 3.97 (s, 3H).

Intermediate 8 2-chloro-6-methoxyquinazoline Step 1: Synthesis of6-methoxyquinazoline-2,4(1H,3H)-dione

A mixture of 2-amino-5-methoxybenzoic acid (5.00 g, 29.9 mmol) and urea(5.40 g, 90.0 mmol) was heated to 200° C. for 1 hour. The mixture wascooled to room temperature and diluted with H₂O (50 mL), the mixture wasfiltered off and the precipitate was washed with H₂O (10 mL), dried invacuo to give the product (5.10 g, yield 88%).

Step 2: Synthesis of 2,4-dichloro-6-methoxyquinazoline

To a solution of 6-methoxyquinazoline-2,4(1H,3H)-dione (5.10 g, 26.6mmol) in POCl₃ (30 mL) was added N,N-diethylaniline (2.4 mL, 15.0 mmol).The mixture was refluxed for 6 hours. The excess POCl₃ was removed underreduced pressure and the residue was dissolved in EtOAc (100 mL), theorganic layer was washed with sat.NaHCO₃ (30 mL), dried over Na₂SO₄,filtered and concentrated to give the crude product which was purifiedby silica gel column (PE/EtOAc=20/1) to give the product (1.70 g, yield28%).

Step 3: Synthesis of 2-chloro-6-methoxyquinazoline

To a solution of 2,4-dichloro-6-methoxyquinazoline (400 mg, 1.75 mmol)in CH₂Cl₂ (10 mL) was added brine (10 mL) containing 9% NH₃.H₂O. Thenzinc powder (336 mg, 5.25 mmol) was added and the mixture was refluxedfor 2 hours. The mixture was diluted with CH₂Cl₂ (50 mL) and filteredoff, the filtrate was neutralized by 2M HCl to pH 7. The aqueous layerextracted with CH₂Cl₂ (20 mL×2), the combined organic layer washed withbrine (30 mL), dried over Na₂SO₄, filtered and concentrated to give thecrude product, which was purified by silica gel column (PE/EtOAc=5/1) togive Intermediate 8 (232 mg, yield 68%). ¹H NMR (CDCl3 300 MHz): δ 9.20(s, 1H), 7.90 (d, J=9.3 Hz, 1H), 7.40 (dd, J=9.3, 2.7 Hz, 1H), 7.15 (d,J=3.0 Hz, 1H), 3.95 (s, 3H).

Intermediate 9 2-chloro-6-methoxy-4-methylquinazoline

A mixture of 2,4-dichloro-6-methoxyquinazoline (synthesis described insteps 1 and 2 of Intermediate 8) (900 mg, 3.93 mmol), MeZnCl (2.95 mL,5.90 mmol, 2M in THF) and Pd(PPh₃)₄ (45.0 mg, 0.0393 mmol) in anhydrousTHF (15 mL) was stirred at 60° C. under N₂ atmosphere for 16 hours.After cooling to 25° C., the mixture was quenched with saturated aqueousNH₄Cl (30 mL), extracted with EtOAc (30 mL×3). The combined organiclayers were washed with H₂O (30 mL) and brine (30 mL), dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure togive the crude product. The crude product was purified by silica gelcolumn (PE/EtOAc=30/1 to 20/1) to give Intermediate 9 (370 mg, yield45%). Structure was confirmed by NOE. ¹H NMR (CDCl₃ 400 MHz): δ 7.88 (d,J=9.2 Hz, 1H), 7.55 (dd, J=9.2, 2.8 Hz, 1H), 7.23 (d, J=2.8 Hz, 1H),3.97 (s, 3H), 2.92 (s, 3H).

Intermediate 10 2-chloroquinazolin-6-ol

To a solution of 2-chloro-6-methoxyquinazoline (Intermediate 8, 260 mg,1.34 mmol) in anhydrous DCM (10 mL) was added AlCl₃ (536 mg, 4.02 mmol).The resulting mixture was stirred at 60° C. for 16 hours. The reactionmixture was cooled to room temperature (26-32° C.) and quenched with icewater (30 mL). The aqueous layer was extracted with DCM (20 mL×3). Thecombined organic layers were washed with brine (20 mL), dried overanhydrous Na₂SO₄, filtered and concentrated to give Intermediate 10 (200mg, yield 83%). ¹H NMR (DMSO-d₆ 400 MHz): δ 10.63 (brs, 1H), 9.41 (s,1H), 7.85 (d, J=9.2 Hz, 1H), 7.62 (dd, J=8.8, 2.4 Hz, 1H), 7.36 (d,J=2.4 Hz, 1H).

Intermediate 11 2-chloro-6-methoxy-3-(trifluoromethyl)quinoxaline Step1: Synthesis of 6-methoxy-3-(trifluoromethyl)quinoxalin-2(1H)-one

A mixture of 4-methoxybenzene-1,2-diamine (2.00 g, 14.5 mmol) and ethyltrifluoropyruvate (2.47 g, 14.5 mmol) in absolute EtOH (30 mL) wasrefluxed overnight. The mixture was cooled to room temperature anddiluted with EtOAc (300 mL), washed with water (100 mL x 2), dried overNa₂SO₄ and concentrated in vacuo. The residue was purified by columnchromatography on silica gel to give6-methoxy-3-(trifluoromethyl)quinoxalin-2(1H)-one (1.50 g, yield 42%) assolid (also obtained 7-methoxy-3-(trifluoromethyl)quinoxalin-2(1H)-one(600 mg, yield 17%)).

Step 2: Synthesis of 2-chloro-6-methoxy-3-(trifluoromethyl)quinoxaline

A mixture of 6-methoxy-3-(trifluoromethyl)quinoxalin-2(1H)-one (500 mg,2.05 mmol) and POCl₃ (4 mL) was stirred under microwave at 140° C. for 2hours. The reaction was quenched with aqueous saturated NaHCO₃ (50 mL)and extracted with EtOAc (50 mL×3), dried over Na₂SO₄ and concentratedin vacuo. The residue was purified by column chromatography on silicagel (PE/EtOAc=10/1) to give Intermediate 11 (460 mg, yield 86%). ¹H NMR(DMSO-d₆ 400 MHz): δ 8.10 (d, J=9.6 Hz, 1H), 7.76 (dd, J=9.2, 2.8 Hz,1H), 7.68 (d, J=2.8 Hz, 1H), 4.01 (s, 3H).

Intermediate 12 3-chloro-7-methoxyisoquinoline Step 1: Synthesis of(E)-2-(hydroxyimino)-6-methoxy-2,3-dihydro-1H-inden-1-one

6-methoxy-2,3-dihydro-1H-inden-1-one (17.2 g, 106 mmol) was dissolved inmethanol (700 mL). The mixture was heated to 40° C., then isoamylnitrite (30 mL, 220 mmol) and concentrated hydrochloric acid (17 mL)were added. The reaction mixture was stirred at 40° C. for 2 hours.After cooled to room temperature, the precipitate was collected byfiltration and dried under high vacuum to obtain product (10.0 g, yield50%).

Step 2: Synthesis of 1,3-dichloro-7-methoxyisoquinoline

To a suspension of(E)-2-(hydroxyimino)-6-methoxy-2,3-dihydro-1H-inden-1-one (3.60 g, 20.0mmol) in POCl₃ (120 mL), PCl₅ (6.50 g, 31.3 mmol) was added at 0° C.,then HCl gas was induced until the solution was saturated. The reactionmixture was stirred at 30° C. for 2 hours. The solvent was removed invacuo and ice water was added to the residue. The precipitate wascollected by filtration, washed with water (30 mL), and dried under highvacuum to give the product (4.50 g, yield 85%) as a solid.

Step 3: Synthesis of 3-chloro-7-methoxyisoquinoline

1,3-dichloro-7-methoxyisoquinoline (1.14 g, 5.00 mmol) was suspended inglacial acetic acid (6 mL) and concentrated HCl (2 mL), then treatedwith tin powder (1.77 g, 15.0 mmol) and stirred at 60° C. for 3 hours.The resulting mixture was basified to pH=9 with concentrated NH₄OH andthen extracted with ethyl acetate (10 mL×3). The combined organic layerwas washed with saturated NaHCO₃ solution (20 mL), dried over Na₂SO₄ andconcentrated in vacuo, followed by silica gel column (EtOAc/PE=20:1) togive Intermediate 12 (200 mg, yield 21%) as a solid. ¹H NMR (DMSO 300MHz): δ 9.00 (s, 1H), 7.94 (s, 1H), 8.87 (d, J=9.0 Hz, 1H), 7.53 (d,J=2.7 Hz, 1H), 7.46 (dd, J=9.0, 2.7 Hz, 1H), 3.89 (s, 3H).

Intermediate 13 3-bromo-7-methoxyquinoline

To a solution of bromo-malonaldehyde (10 g, 66.2 mmol) in absoluteethanol (100 mL) was added 3-methoxyaniline (7.3 g, 59.6 mmol). Themixture was heated to 30° C. and stirred at that temperature for 24 h.Conc. HCl (100 mL) was added, and the reaction mixture was heated at100° C. for two days. After the mixture was concentrated and the residuewas partitioned between ethyl acetate (150 mL) and saturated sodiumcarbonate (50 mL). The organic phase was washed with brine, dried withanhydrous Na₂SO₄ and concentrated to give a black oil, which waspurified by column chromatography (PE/EA=3/1) to give Intermediate 13 asa brown oil (500 mg, 3.8%). MS (ESI): m/z 237.9 [M+1]⁺.

Intermediate 14 2-chloro-3-(trifluoromethyl)quinoxalin-6-ol

Followed the procedure described for preparing Intermediate 10 startingwith 2-chloro-6-methoxy-3-(trifluoromethyl)quinoxaline (Intermediate11), with a purification by silica gel column chromatography.

Intermediate 15 3-chloro-7-methoxy-4-methylisoquinoline

Step 1: To a solution of i-Pr₂NH (1.95 g, 19.3 mmol) in anhydrous THF(40 mL) was added dropwise n-BuLi (7.72 mL, 19.3 mmol, 2.50 M in hexane)at −78° C. under N₂ atmosphere. After 30 minutes,1,3-dichloro-7-methoxyisoquinoline (described in step 2 of Intermediate12) (4.00 g, 17.5 mmol) in anhydrous THF (20 mL) was added to thereaction. After 15 minutes, MeI (4.97 g, 35.0 mmol) was added dropwise.The resulting mixture was warmed to 25° C. and allowed to stir for 16hours. The reaction was quenched with H₂O (150 mL) followed by an ethylacetate extraction/workup to give the crude product, which was purifiedby prep-HPLC (0.1% TFA as additive). Most of CH₃CN was removed underreduced pressure and extracted with EtOAc (50 mL×3). The combinedorganic layers were washed with H₂O (50 mL) and brine (50 mL), driedover anhydrous Na₂SO₄, filtered and concentrated in vacuo to give1,3-dichloro-7-methoxy-4-methylisoquinoline (2.0 g, yield 47%) as anoff-white solid.

Step 2: To a solution of the above product (1.00 g, 4.13 mmol) inCH₃COOH (18 mL) and HCl (6 mL, 36%) was added Sn (1.46 g, 12.4 mmol) at25-30° C. The resulting mixture was stirred at 60° C. for 2.5 hours. Thereaction mixture was basified with aqueous NaOH (2M) to pH=7˜8, and theresulting mixture was extracted with EtOAc, followed by purification viasilica gel column (PE/EtOAc=50/1 to 10/1) to give Intermediate 15 (470mg, yield 55%) as a solid. ¹H NMR (CDCl₃ 400 MHz): δ 8.82 (s, 1H), 7.89(d, J=9.6 Hz, 1H), 7.39 (dd, J=9.6, 2.8 Hz, 1H), 7.19 (d, J=2.8 Hz, 1H),3.95 (s, 3H), 2.69 (s, 3H).

Intermediate 16 2-chloro-5-fluoroquinolin-6-ol

To a solution of 2-chloroquinolin-6-ol (Intermediate 2, 1.8 g, 85% pure,8.5 mmol) in CH₃CN (50 mL) was added Selectfluor (4.20 g, 12.0 mmol).The reaction mixture was heated at 60° C. overnight and concentrated.The residue was partitioned between water (50 mL) and EtOAc (100 mL).The aqueous phase was separated and extracted with ethyl acetate. Thecombined organic layers were washed with brine, dried over Na₂SO₄,filtered and concentrated. The residue was purified by silica gelchromatography to give 2-chloro-5-fluoroquinolin-6-ol using ethylacetate and hexanes as solvents (0.4 g, 21% yield).

Example 73 GSNOR Assays

Various compounds were tested in vitro for their ability to inhibitGSNOR activity. GSNOR inhibitor compounds in Examples 1-71 had an IC₅₀of about <10 μM. GSNOR inhibitor compounds in Examples 1-4, 6, 8, 10-14,16-35, 37-43, 45, 47-50, 52-62, 65-68, 70-71 had an IC₅₀ of about <0.5μM. GSNOR inhibitor compounds in Examples 1-4, 8, 10-14, 17-28, 30, 31,37, 40-41, 43, 45, 47-50, 52-58, 60-61, 65, 67-68, and 70-71 had an IC₅₀of about <0.1 μM.

GSNOR expression and purification is described in Biochemistry 2000, 39,10720-10729.

GSNOR Fermentation:

Pre-cultures were grown from stabs of a GSNOR glycerol stock in 2XYTmedia containing 100 ug/ml ampicillin after an overnight incubation at37° C. Cells were then added to fresh 2XYT (4 L) containing ampicillinand grown to an OD (A₆₀₀) of 0.6-0.9 at 37° C. before induction. GSNORexpression was induced with 0.1% arabinose in an overnight incubation at20° C.

GSNOR Purification:

E. coli cell paste was lysed by nitrogen cavitation and the clarifiedlysate purified by Ni affinity chromatography on an AKTA FPLC (AmershamPharmacia). The column was eluted in 20 mM Tris pH 8.0/250 mM NaCl witha 0-500 mM imidazole gradient. Eluted GSNOR fractions containing theSmt-GSNOR fusion were digested overnight with Ulp-1 at 4° C. to removethe affinity tag then re-run on the Ni column under the same conditions.GSNOR was recovered in the flowthrough fraction and for crystallographyis further purified by Q-Sepharose and Heparin flowthroughchromatography in 20 mM Tris pH 8.0, 1 mM DTT, 10 uM ZnSO₄.

GSNOR Assay:

GSNO and enzyme/NADH Solutions are made up fresh each day. The solutionsare filtered and allowed to warm to room temperature. GSNO solution: 100mM NaPO4 (pH 7.4), 0.480 mM GSNO. 396 μL of GSNO Solution is added to acuvette followed by 8 μL of test compound in DMSO (or DMSO only for fullreaction control) and mixed with the pipette tip. Compounds to be testedare made up at a stock concentration of 10 mM in 100% DMSO. 2 foldserial dilutions are done in 100% DMSO. 8 μL of each dilution are addedto an assay so that the final concentration of DMSO in the assay is 1%.The concentrations of compounds tested range from 100 to 0.003 μM.Enzyme/NADH solution: 100 mM NaPO₄ (pH 7.4), 0.600 mM NADH, 1.0 μg/mLGSNO Reductase. 396 μL of the Enzyme/NADH solution is added to thecuvette to start the reaction. The cuvette is placed in the Cary 3EUV/Visible Spectrophotometer and the change in 340 nm absorbance/min at25° C. is recorded for 3 minutes. The assays are done in triplicate foreach compound concentration. IC₅₀'s for each compound are calculatedusing the standard curve analysis in the Enzyme Kinetics Module ofSigmaPlot.

Final assay conditions: 100 mM NaPO₄, pH 7.4, 0.240 mM GSNO, 0.300 mMNADH, 0.5 μg/mL GSNO Reductase, and 1% DMSO. Final volume: 800μL/cuvette.

Example 74 Efficacy of GSNORi in Experimental Asthma

Experimental Asthma Model:

A mouse model of ovalbumin (OVA)-induced asthma was used to screen GSNORinhibitors for efficacy against methacholine (MCh)-inducedbronchoconstriction/airway hyper-responsiveness. This is a widely usedand well characterized model that presents with an acute, allergicasthma phenotype with similarities to human asthma. Efficacy of GSNORinhibitors was assessed using a protocol in which GSNOR inhibitors wereadministered after OVA sensitization and airway challenge, and prior tochallenge with MCh. Bronchoconstriction in response to challenge withincreasing doses of MCh was assessed using whole body plethysmography(P_(enh); Buxco). The amount of eosinophil infiltrate into thebronchoaveolar lavage fluid (BALF) was also determined as a measure oflung inflammation. The effects of GSNOR inhibitors were compared tovehicles and to Combivent (inhaled; IH) as the positive control.

Materials and Method

Allergen Sensitization and Challenge Protocol

OVA (500 μg/ml) in PBS was mixed with equal volumes of 10% (w/v)aluminum potassium sulfate in distilled water and incubated for 60 min.at room temperature after adjustment to pH 6.5 using 10 N NaOH. Aftercentrifugation at 750×g for 5 min, the OVA/alum pellet was resuspendedto the original volume in distilled water. Mice received anintraperitoneal (IP) injection of 100 μg OVA (0.2 mL of 500 μg/mL innormal saline) complexed with alum on day 0. Mice were anesthetized byIP injection of a 0.2-mL mixture of ketamine and xylazine (0.44 and 6.3mg/mL, respectively) in normal saline and were placed on a board in thesupine position. Two hundred fifty micrograms (100 μl of a 2.5 mg/ml) ofOVA (on day 8) and 125 μg (50 μl of 2.5 mg/ml) OVA (on days 15, 18, and21) were placed on the back of the tongue of each animal.

Pulmonary Function Testing (Penh)

In vivo airway responsiveness to methacholine was measured 24 h afterthe last OVA challenge in conscious, freely moving, spontaneouslybreathing mice with whole body plethysmography using a Buxco chamber(Wilmington, N.C.). Mice were challenged with aerosolized saline orincreasing doses of methacholine (5, 20, and 50 mg/mL) generated by anultrasonic nebulizer for 2 min. The degree of bronchoconstriction wasexpressed as enhanced pause (P_(enh)), a calculated dimensionless value,which correlates with the measurement of airway resistance, impedance,and intrapleural pressure in the same mouse. P_(enh) readings were takenand averaged for 4 min. after each nebulization challenge. P_(enh) iscalculated as follows: P_(enh)=[(T_(e)/T_(r)−1)×(PEF/PIF)], where T_(e)is expiration time, T_(r) is relaxation time, PEF is peak expiratoryflow, and PIF is peak inspiratory flow×0.67 coefficient. The time forthe box pressure to change from a maximum to a user-defined percentageof the maximum represents the relaxation time. The T_(r) measurementbegins at the maximum box pressure and ends at 40%.

Eosinophil Infiltrate in BALF

After measurement of airway hyper-reactivity, the mice wereexsanguinated by cardiac puncture, and then BALF was collected fromeither both lungs or from the right lung after tying off the left lungat the mainstem bronchus. Total BALF cells were counted from a 0.05 mLaliquot, and the remaining fluid was centrifuged at 200×g for 10 min at4° C. Cell pellets were resuspended in saline containing 10% BSA withsmears made on glass slides. Eosinophils were stained for 5 min. with0.05% aqueous eosin and 5% acetone in distilled water, rinsed withdistilled water, and counterstained with 0.07% methylene blue.Alternatively, eosinophils and other leukocytes were stained withDiffQuik.

GSNOR Inhibitors and Controls

GSNOR inhibitors were reconstituted in phosphate buffered saline (PBS),pH 7.4, or 0.5% w/v carboxy methylcellulose at concentrations rangingfrom 0.00005 to 3 mg/mL. GSNOR inhibitors were administered to mice (10mL/kg) as a single dose or multiple dose either intravenously (IV) ororally via gavage. Dosing was performed from 30 min. to 72 h prior toMCh challenge. Effects of GSNOR inhibitors were compared to vehicledosed in the same manner.

Combivent was used as the positive control in all studies. Combivent(Boehringer Ingelheim) was administered to the lung using the inhalerdevice supplied with the product, but adapted for administration tomice, using a pipette tip. Combivent was administered 48 h, 24 h, and 1h prior to MCh challenge. Each puff (or dose) of Combivent provides adose of 18 μg ipatropium bromide (IpBr) and 103 μg albuterol sulfate orapproximately 0.9 mg/kg IpBr and 5 mg/kg albuterol.

Statistical Analyses

Area under the curve values for P_(enh) across baseline, saline, andincreasing doses of MCh challenge were calculated using GraphPad Prism5.0 (San Diego, Calif.) and expressed as a percent of the respective (IVor orally administered) vehicle control. Statistical differences amongtreatment groups and the respective vehicle control group within eachstudy were calculated using one-way ANOVA, Dunnetts or Bonferronipost-hoc tests or t-test (JMP 8.0, SAS Institute, Cary, N.C. orMicrosoft Excel). A p value of <0.05 among the treatment groups and therespective vehicle control group was considered significantly different.

Results

In the OVA model of asthma, the compound of Example 8 significantly(p<0.05) decreased eosinophil infiltration in BAL by 37% of vehiclecontrol when given via three oral doses of 10 mg/kg at 48 h, 24 h, and 1h prior to assessment.

In the OVA model of asthma, the compound of Example 3 significantly(p<0.05) decreased eosinophil infiltration in BAL by 42% of vehiclecontrol when given via three oral doses of 10 mg/kg at 48 h, 24 h, and 1h prior to assessment.

In the OVA model of asthma, the compound of Example 27 significantly(p<0.05) decreased eosinophil infiltration in BAL by 23% of vehiclecontrol when given via three oral doses of 10 mg/kg at 48 h, 24 h, and 1h prior to assessment.

In the OVA model of asthma, the compound of Example 4 significantly(p<0.05) decreased MCh-induced AHR by 19% of vehicle control anddecreased eosiinophil infiltration into BALF by 20% of vehicle controlwhen given as a single IV dose of 10 mg/kg at 24 h prior to assessment.

Example 75 Mouse Pharmacokinetic (PK) Study

Experimental Model

The mouse was used to determine the pharmacokinetics of compounds of theinvention. This species is widely used to assess the bioavailability ofcompounds by administering both oral (PO) and intravenous (IV) testarticles. Efficacy of the compounds of the invention was compared byassessing plasma exposure in male BALB/c mice either via IV or POadministration at the times of peak activity.

Materials and Methods

IV Administration of Compounds of the Invention

Compounds of the invention were reconstituted in a phosphate bufferedsaline (PBS)/10% Solutol (HS 15) clear solution resulting in aconcentration of 0.2 mg/mL and administered to mice (2 mg/kg) as asingle IV dose. Animals were dosed via the lateral tail vein. Bloodsamples were collected at designated time points (0.083, 0.25, 0.5, 1,2, 4, 8, 16, 24 hours) by cardiac puncture under isoflurane anesthesia(up to 1 mL blood per animal). The blood was collected into tubescontaining Li-Heparin. The blood samples were kept on ice untilcentrifugation within approximately 30 minutes of collection. The plasmawas transferred into labeled polypropylene tubes and frozen at −70° C.until analyzed by LC/MS/MS.

PO Administration of Compounds of the Invention

The compounds of the invention were reconstituted in 40% PropyleneGlycol/40% Propylene Carbonate/20% of a 5% Sucrose clear solutionresulting in a concentration of 2 mg/mL and administered to mice (10mg/kg) as a single oral dose via gavage. Blood samples were collected at0.25, 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 hours post dose by cardiacpuncture under isoflurane anesthesia. The blood was collected in tubescontaining Li-Heparin. The blood samples were kept on ice untilcentrifugation within approximately 30 minutes of collection. The plasmawas transferred into labeled polypropylene tubes and frozen at −70° C.until analyzed by LC/MS/MS.

LC/MS/MS Analysis

Plasma samples at each timepoint were analyzed using a LC-MS/MS with alower limit of quantification (LLOQ) of 1 ng/mL. Plasma was analyzed todetermine the amount of the compound of the invention in each sample andregression curves generated for each compounds of the invention in therelevant matrixes.

WinNonlin analysis was used for calculating PK parameters for both theIV and PO administrations:

PK parameters for IV portion—AUC_(last); AUC_(INF); T1/2; Cl; Vss;C_(max); MRT

PK parameters for PO portion—AUC_(last); AUC_(INF); T1/2; C_(max); Cl,MRT.

In addition to the above PK parameters, bioavailability (% F) wascalculated.

Compounds in Examples 3, 4, 8, 13, 19, 27, and 28 were tested and allhad an oral bioavailability of greater than 9%. Compounds in Examples 3,8, 13, and 27 had an oral bioavailability of greater than 45%.

Example 76 Efficacy of GSNOR Inhibitors in Experimental InflammatoryBowel Disease (IBD)

Overview of the Models:

Acute and chronic models of dextran sodium sulfate (DSS)-induced IBD inmice were used to explore efficacy of GSNORi against this disease. Acuteand chronic DSS-induced IBD are widely used and well characterizedmodels that induce pathological changes in the colon similar to thoseobserved in the human disease. In these models and in human disease,epithelial cells within the crypts of the colon are disrupted, leadingto dysfunction of the epithelial barrier and the ensuing tissueinflammation, edema, and ulceration. GSNORi therapy may benefit IBD byrestoring s-nitrosoglutathione (GSNO) levels, and thus prevent orreverse the epithelial barrier dysfunction.

Acute Prophylactic Model:

Experimental IBD was induced by administration of DSS in the drinkingwater of male C57Bl/6 mice (N=8 to 10 mice per group) for 6 consecutivedays. GSNORi was dosed orally at doses of 0.1 to 10 mg/kg/day for 10days starting two days prior to and continuing two days post DSSexposure. Two days post DSS exposure, the effect of GSNORi was assessedin a blinded fashion via endoscopy and histopathology using a five pointscale ranging from a score=0 (normal tissue) through a score=4(ulcerative tissue damage and marked pathological changes). Levels ofcirculating cytokines involved in inflammatory pathways were alsoassessed. The effect of GSNORi was compared to vehicle treated controls.The corticosteroid, prednisolone, was used as the positive control inthis study and was administered daily at 3 mg/kg/day via oral dosing.Naïve mice (N=5) were also assessed as a normal tissue control.

Chronic Treatment Model:

Experimental IBD was induced by administration of DSS in the drinkingwater of male C57Bl/6 mice (N=10 to 12 mice per group) for 6 consecutivedays. GSNORi was dosed orally at doses of 10 mg/kg/day for 14 daysstarting one day after cessation of DSS exposure. Efficacy of GSNORi wasassessed in a blinded fashion via endoscopy after 7 days and 14 days ofGSNORi dosing and via histopathology after 14 days of GSNORi dosingusing a five point scale ranging from a score=0 (normal tissue) througha score=4 (ulcerative tissue damage and marked pathological changes).Levels of circulating cytokines involved in inflammatory pathways werealso assessed. The effect of GSNORi was compared to vehicle treatedcontrols. The corticosteroid, prednisolone, was used as the positivecontrol in this study and was administered daily at 3 mg/kg/day via oraldosing. Naïve mice (N=5) were also assessed as a normal tissue control.

Results:

The compound of Example 3 attenuated colon injury and lowered levels ofcytokines involved in inflammatory responses in a mouse model of acuteDSS-induced IBD. The percent of mice presenting with severe colon injuryscores via endoscopy and histopathology assessments was significantly(p<0.05) decreased by 38% to 88% of vehicle control after oral treatmentwith the compound of Example 3 at 0.1, 1, or 10 mg/kg/day for 10consecutive days using a prophylactic dosing regimen. The compound ofExample 3 also restored circulating inflammatory cytokines towardslevels observed in untreated naïve mice. The effects of the compound ofExample 3 were comparable to or greater than those observed forprednisolone.

The compound of Example 8 attenuated colon injury in a mouse model ofacute DSS-induced IBD. The percent of mice presenting with severe coloninjury scores via endoscopy or histopathology assessments was decreasedby 44% or 26%, respectively, of vehicle control after oral treatmentwith the compound of Example 8 at 10 mg/kg/day for 10 consecutive daysusing a prophylactic dosing regimen.

The compound of Example 19 attenuated colon injury in a mouse model ofacute DSS-induced IBD. The percent of mice presenting with severe coloninjury scores via endoscopy assessment was decreased by 31% of vehiclecontrol after oral treatment with the compound of Example 19 at 10mg/kg/day for 10 consecutive days using a prophylactic dosing regimen.

The compound of Example 13 attenuated colon injury in a mouse model ofchronic DSS-induced IBD. The percent of mice presenting with severecolon injury scores via endoscopy or histopathology assessment wassignificantly (p<0.05) decreased by 52% or 53%, respectively, of vehiclecontrol after oral treatment with the compound of Example 13 at 10mg/kg/day for up to 14 consecutive days using a treatment dosingregimen.

Example 77 Efficacy of GSNOR Inhibitors in Experimental ChronicObstructive Pulmonary Disease (COPD)

Short Duration Cigarette Smoke COPD Models

The efficacy of GSNOR inhibitors was assessed in a mouse model ofchronic obstructive pulmonary disease (COPD) induced by short duration(4 days or 11 days) of exposure to cigarette smoke. Infiltration ofinflammatory cells into the bronchoalveolar lavage fluid (BALF) and BALFlevels of chemokines involved in inflammation and tissue turnover/repairwere measured to assess the influences of GSNOR inhibitors on some ofthe early events associated with the initiation and progression of COPD.

Overview of the Models:

Efficacy of GSNOR inhibitors against COPD was explored using acute (4day) and subchronic (11 day) models of cigarette smoke-induced COPD inmice. Exposure of animals to cigarette smoke provides a model of COPD inwhich injury is induced by the same causative agent as in human diseaseand in which injury exhibits similarities to the human disease,including airway obstruction, airspace enlargement, and involvement ofinflammatory responses in these pathologies. In animal models, changesin lung pathology are only evident after extended (several months)duration of exposure to cigarette smoke, thus making chronic modelsprohibitive as effective screening tools. More recently, modelsexploring inflammatory responses after short duration (2 weeks or less)of smoke exposure in mice have been utilized as tools for screeningefficacy and mechanisms of action of novel therapeutics against COPD.The key roles of inflammation in the initiation and progression of COPD,make these short duration models relevant for initial tests of efficacyof novel therapeutics.

Acute (4 Day) Smoke Exposure Model:

Female C57Bl/6 mice (N=8 per group) were exposed to cigarette smokeusing a whole body exposure chamber. Mice were exposed daily for 4consecutive days to 4 cycles of smoke from 6 sequential cigarettes(Kentucky 3R4F without filter) with a 30 minute smoke free intervalbetween cycles. GSNOR inhibitors were administered daily via oral dosingat 10 mg/kg/day for 7 days starting 2 days prior to smoke exposure andcontinuing 1 day post-exposure. The effects of GSNOR inhibitors wereassessed by quantitating the numbers of total cells, leukocytes, andleukocytes differentials in the BALF via light microscopy and the levelsof BALF chemokines via ELISA at approximately 24 h after the last smokeexposure. The effect of GSNOR inhibitors were compared to vehicletreated controls. The PDE4 inhibitor, roflumilast, was used as thepositive control for the study. A group of naïve mice (N=8) was exposedto air and used as a negative control for the study.

Subchronic (11 Day) Smoke Exposure Model:

Female C57Bl/6 mice (N=10 per group) were exposed to cigarette smokegenerated from Marlboro 100 cigarettes without filters. Exposure timeswere 25 min. on study day 1, 35 min. on study day 2, and 45 min. onstudy days 3 to 11. GSNOR inhibitors were administered one hour prior tosmoke exposure on each day. GSNOR inhibitors were dosed orally at 1 to10 mg/kg/day for 11 days. The effects of GSNOR inhibitors were assessedby quantitating the number of total cells, and leukocytes differentialsin the BALF via light microscopy at 24 h after the last exposure. Theeffect of GSNOR inhibitors were compared to vehicle treated controls andexpressed as percent inhibition of the cigarette smoke induced increasesin BALF cell numbers. Roflumilast was used as the positive control forthe study and was dosed at 5 mg/kg/day. A group of naïve mice (N=10) wasexposed to air and dosed with vehicle as a negative control for thestudy.

Results:

The compound of Example 3 attenuated the smoke-induced changes in BALFcellular infiltrate and BALF inflammatory chemokines. The compound ofExample 3 significantly (p<0.05) decreased total cells, leukocytes,macrophages, neutrophils, and eosinophils in BALF by 66%, 80%, 75%, 84%,and 95%, respectively, compared to vehicle treated controls when dosedorally at 10 mg/kg/day for 7 days in the acute 4 day smoke model. Theseeffects of the compound of Example 3 were comparable to or greater thanthose observed for roflumilast. The compound of Example 3 also restoredBALF chemokines towards levels observed in naïve mice. In the subchronic11 day model, the compound of Example 3 inhibited the smoke-inducedincrease in total cells (p<0.05), macrophages, neutrophils (p<0.05),eosinophils, and lymphocytes (p<0.05) in BALF by 25%, 24%, 41%, 70%, and49%, respectively, when dosed orally at 10 mg/kg/day for 11 days. Whendosed orally at 5 mg/kg/day, the compound of Example 3 inhibited totalcells, macrophages, neutrophils (p<0.05), and lymphocytes (p<0.05) inBALF by 22%, 23%, 29%, and 46%, respectively.

The compound of Example 8 significantly (p<0.05) inhibited thesmoke-induced increase in total cells, macrophages, neutrophils, andlymphocytes in BAL by 35% to 48%, 24% to 43%, 41% to 70%, and 49 to 65%,respectively, when dosed orally at 1 to 10 mg/kg/day for 11 days in thesubchronic 11 day model. There was no dose response of effect with 1, 5,or 10 mg/kg/day. The effects of the compound of Example 8 werecomparable to those of roflumilast.

The compound of Example 13 significantly (p<0.05) inhibited thesmoke-induced increase in total cells, macrophages, neutrophils, andlymphocytes in BAL by 56%, 53%, 67%, and 60%, respectively, when dosedorally at 1 mg/kg/day for 11 days in the subchronic 11 day model. Theseeffects of the compound of Example 13 were comparable to those ofroflumilast.

The compound of Example 27 significantly (p<0.05) inhibited thesmoke-induced increase in total cells, macrophages, neutrophils, andlymphocytes in BAL by 44%, 41%, 64%, and 46%, respectively, when dosedorally at 1 mg/kg/day for 11 days in the subchronic 11 day model. Theseeffects of the compound of Example 27 were comparable to those ofroflumilast.

Example 78 An Exploratory Mouse Study of Acetaminophen Toxicity

S-nitrosoglutathione reductase (GSNOR) inhibition has been previouslyshown in our hands to ameliorate the negative manifestations ofgastrointestinal injury in animal models. As an extension of theseobservations, the effects of S-nitrosoglutathione (GSNO) or GSNORinhibitors (GSNORi) on acetaminophen (ACAP) induced liver toxicity canbe evaluated in a mouse model of liver injury. Blood samples arecollected for liver function assays and tissue samples are collected atthe end of the study for histopathologic examination.

Materials and Methods

GSNORi, GSNO, acetaminophen (ACAP, Sigma) Vehicles (1/2 cc syringes fordosing), Isoflurane, 18 1 cc syringes w/26 g needles for bloodcollection, 90 serum separator tubes for clinical chemistry.

General Study Design:

Animals (5/group) are acclimated for at least 3 days prior to dosing. OnStudy Day 1, acetaminophen treatment (300 mg/kg PO) was given a singletime=0 to fasted animals. Two hours later, GSNORi (10 mg/kg/dose) orGSNO (5 mg/kg/dose) are intravenously administered to the treatmentgroups. GSNORi or GSNO are given at 24 and 48 hours-post their initialadministration to the treatment groups. Mice are observed for signs ofclinical toxicity and blood was collected at 6, 24, and 72 hourspost-ACAP administration for liver function tests: Alkaline phosphatase(ALK); Alanine aminotransferase (ALT); Aspartate aminotransferase (AST);Gamma glutamyltransferase (GGT) and Total bilirubin (TBILI). Livers arecollected at 72 hours for histopathologic examination.

Study Outline

Drug Group Treatment Dose Concentration N 1 ACAP PO 300 mg/kg  10 ml/kg 5 2 Saline PO 0 mg/kg 10 ml/kg  5 3 GSNORi IV 10 mg/kg  1 mg/mL 5 4 GSNOIV 5 mg/kg 1 mg/mL 5 5 GSNORi IV + ACAP 10 m/k/300 m/k 1 mg/mL 5 6 GSNOIV + ACAP  5 m/k/300 m/k 1 mg/mL 5

Study Calendar:

Day −6 Receive mice and place in regular cages Day −1 Fast animalsovernight Day 0 Weigh, PO ACAP time = 0, time = 2 IV GSNO or GSNORibleed all groups at 6 hr post-ACAP Day 1 Weigh, bleed all groups for 24hr LFTs, IV GSNO or GSNORi Day 2 Weigh, IV GSNO or GSNORi Day 3 Bleedfor 72 hr LFTs, collect livers for weight and histology

Vehicle, GSNO and GSNORi Preparation

The vehicle control article is Phosphate Buffered Saline (PBS) (notcontaining calcium, potassium, or magnesium) adjusted to pH 7.4. Thevehicle components are weighed into a container on a tared balance andbrought to volume with purified water (w/v). The 10× stock solution ismixed using a magnetic stirrer, as necessary. Thereafter, the 10× stocksolution is diluted with deionized water at a ratio of 1:9 (v/v). GSNOis warmed to room temperature before preparation of solutions. Prior touse, the PBS solution is nitrogen sparged. 1 mg/mL GSNO solutions arekept cold (i.e., kept on an ice bath) and protected from light and usedwithin 4 hours of preparation. GSNORi Preparation, the 1 mg/mLconcentration is reconstituted in phosphate buffered saline (PBS), pH7.4. GSNORi is administered to mice (10 mL/kg) as a single (IV) dailydose. Dosing is performed 2 hours post-ACAP administration and then 26and 50 hours later. Effects of GSNO or GSNORi are compared to ACAP andsaline vehicle dosed in the same manner.

Calculations:

Mean body weights, mean liver organ weights and clinical pathologyendpoints (+/−SD) with T-test and ANOVA (alpha=0.05) comparison tovehicle control group. The clinical pathology data are prepared as meanvalues unless the data are not normally distributed, in which case,median values can be presented with the minimum and maximum value range.

Example 79 An Exploratory Study to Assess the Anti NASH FibroticActivity of GSNORi in STAM Mice

S-nitrosoglutathione reductase (GSNOR) inhibition has been previouslyshown in our hands to ameliorate the negative manifestations ofgastrointestinal injury and ACAP injury in mouse models. As an extensionof these observations, the effects of GSNOR inhibitors (GSNORi) abilityto reverse fibrotic activity in nonalcoholic steatohepatitis(NASH)-induced liver disease is evaluated in STAM (signal transducingadaptor molecule) mice. In these mice sequential changes are seen fromliver steatosis to fibrosis within two weeks and there are closesimilarities to human NASH histopathology.

Materials and Methods

GSNORi, Telmisartan, Vehicles (1/2 cc syringes for dosing), Isoflurane,18 1 cc syringes w/26 g needles for blood collection, 90 serum separatortubes for clinical chemistry.

General Study Design:

Animals (6/group) are acclimated prior to beginning the Study. At 4weeks of age the animals are put on a diet, group 1 (normal mice)receives a normal diet while groups 2-4 (STAM mice) are put on a highfat diet for the duration of the Study. At Study Week 7 the mice beginoral daily dosing with GSNORi and are sacrificed at Study Week 9. Miceare observed for signs of clinical toxicity and blood/tissue iscollected for liver analyses: Plasma triglycerides (TG); Alanineaminotransferase (ALT); Aspartate aminotransferase (AST); GeneExpression: Timp-1, α-SMA, collagen 3, TNF-α and MCP-1 as well ashistopathologic examination using HE staining for (NAFLD) activity scoreand Sirius-red staining (fibrosis area).

Study Outline

Drug Group Treatment Diet Dose Concentration N 1 normal ND  0 mg/kg 0ml/kg  6 2 STAM + vehicle HFD 10 mg/kg 1 mg/mL 6 3 STAM + GSNORi IV HFD10 mg/kg 1 mg/mL 6 4 STAM + Telmisarten HFD 10 mg/kg 1 mg/mL 6 ND:normal diet, HFD: high fat diet

Calculations:

Mean body weights, mean liver organ weights and clinical pathologyendpoints (+/−SD) with T-test and ANOVA (alpha=0.05) comparison tovehicle control group. The clinical pathology data are prepared as meanvalues unless the data are not normally distributed, in which case,median values were presented with the minimum and maximum value range.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention.

We claim:
 1. The compound of Formula I:

wherein Z₁ is selected from the group consisting of CR_(2a) and N; Z₂ isselected from the group consisting of CR_(2b) and N; Z₃ is selected fromthe group consisting of CR₂ and N; with the proviso that at least 1 ofZ₁, Z₂, or Z₃ must be N; m is selected from the group consisting of 0,1, 2, or 3; R₁ is independently selected from the group consisting ofchloro, fluoro, and bromo; R_(2a), R_(2b), and R_(2c), are independentlyselected from the group consisting of hydrogen, halogen, C₁-C₃ alkyl,fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and N(CH₃)₂; X is selectedfrom the group consisting of

n is selected from 0, 1, and 2; R₃ is independently selected from thegroup consisting of halogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl,cyano, C₁-C₃ alkoxy, and NR₄R_(4′) where R₄ and R_(4′) are independentlyselected from the group consisting of C₁-C₃ alkyl, or R₄ when takentogether with R_(4′) form a ring with 3 to 6 members; A is selected fromthe group consisting of

or a pharmaceutically acceptable salt, stereoisomer, or N-oxide thereof.2. The compound of claim 1 wherein m is selected from the groupconsisting of 0 and 1; R_(2a), R_(2b), and R_(2c), are independentlyselected from the group consisting of hydrogen, chloro, fluoro, methyl,trifluoromethyl, cyano, methoxy, and N(CH₃)₂; n is selected from thegroup consisting of 0 and 1; and R₃ is independently selected from thegroup consisting of fluoro, chloro, methyl, trifluoromethyl, cyano,methoxy, and NR₄R_(4′) where R₄ and R_(4′) are methyl, or alternativelytogether with the said N form the ring aziridin-1-yl or morpholino. 3.The compound of claim 2 wherein X is


4. The compound of claim 3 wherein A is COOH.
 5. The compound of claim1, wherein the compound is a compound of Formula II

wherein Z₂ is selected from the group consisting of CR_(2b) and N; Z₃ isselected from the group consisting of CR_(2c) and N; m is selected fromthe group consisting of 0, 1, 2, or 3; R₁ is independently selected fromthe group consisting of chloro, fluoro, and bromo; R_(2b) and R_(2c) areindependently selected from the group consisting of hydrogen, halogen,C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and N(CH₃)₂;X is selected from the group consisting of

R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members; n is selected from 0, 1, and 2; A is selected from the groupconsisting of

or a pharmaceutically acceptable salt, stereoisomer, or N-oxide thereof.6. The compound of claim 5 wherein m is selected from the groupconsisting of 0 and 1; R_(2a), R_(2b), and R_(2c), are independentlyselected from the group consisting of hydrogen, chloro, fluoro, methyl,trifluoromethyl, cyano, methoxy, and N(CH₃)₂; n is selected from thegroup consisting of 0 and 1; and R₃ is independently selected from thegroup consisting of fluoro, chloro, methyl, trifluoromethyl, cyano,methoxy, and NR₄R_(4′) where R₄ and R_(4′) are methyl, or alternativelytogether with the said N form the ring aziridin-1-yl or morpholino.
 7. Apharmaceutical composition comprising a therapeutically effective amountof a compound according to claim 1 together with a pharmaceuticallyaccepted carrier or excipient.
 8. A method of making a pharmaceuticalcomposition according to claim 7 comprising combining a compound ofFormula I as defined in claim 1 with a pharmaceutically accepted carrieror excipient.
 9. A method of treatment of a disorder which comprisesadministering to a patient in need thereof a therapeutically effectiveamount of a compound of Formula (I):

wherein Z₁ is selected from the group consisting of CR_(2a) and N; Z₂ isselected from the group consisting of CR_(2b) and N; Z₃ is selected fromthe group consisting of CR_(2c) and N; with the proviso that at least 1of Z₁, Z₂, or Z₃ must be N; m is selected from the group consisting of0, 1, 2, or 3; R₁ is independently selected from the group consisting ofchloro, fluoro, and bromo; R_(2a), R_(2b), and R_(2c), are independentlyselected from the group consisting of hydrogen, halogen, C₁-C₃ alkyl,fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and N(CH₃)₂; X is selectedfrom the group consisting of

n is selected from 0, 1, and 2; R₃ is independently selected from thegroup consisting of halogen, C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl,cyano, C₁-C₃ alkoxy, and NR₄R_(4′) where R₄ and R_(4′) are independentlyselected from the group consisting of C₁-C₃ alkyl, or R₄ when takentogether with R_(4′) form a ring with 3 to 6 members; A is selected fromthe group consisting of

or a pharmaceutically acceptable salt, stereoisomer, or N-oxide thereof;wherein the disorder is selected from the group consisting of pulmonarydisorders associated with hypoxemia and/or smooth muscle constriction inthe lungs and airways and/or lung infection and/or lung inflammationand/or lung injury, inflammatory diseases and functional boweldisorders.
 10. The compound of claim 9 wherein m is selected from thegroup consisting of 0 and 1; R_(2a), R_(2b), and R_(2c), areindependently selected from the group consisting of hydrogen, chloro,fluoro, methyl, trifluoromethyl, cyano, methoxy, and N(CH₃)₂; n isselected from the group consisting of 0 and 1; and R₃ is independentlyselected from the group consisting of fluoro, chloro, methyl,trifluoromethyl, cyano, methoxy, and NR₄R_(4′) where R₄ and R_(4′) aremethyl, or alternatively together with the said N form the ringaziridin-1-yl or morpholino.
 11. The compound of claim 10 wherein X is


12. The compound of claim 11 wherein A is COOH.
 13. The compound ofclaim 9, wherein the compound is a compound of Formula II

wherein Z₂ is selected from the group consisting of CR_(2b) and N; Z₃ isselected from the group consisting of CR_(2c) and N; m is selected fromthe group consisting of 0, 1, 2, or 3; R₁ is independently selected fromthe group consisting of chloro, fluoro, and bromo; R_(2b) and R_(2c) areindependently selected from the group consisting of hydrogen, halogen,C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and N(CH₃)₂;X is selected from the group consisting of

R₃ is independently selected from the group consisting of halogen, C₁-C₃alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and NR₄R_(4′) whereR₄ and R_(4′) are independently selected from the group consisting ofC₁-C₃ alkyl, or R₄ when taken together with R_(4′) form a ring with 3 to6 members; n is selected from 0, 1, and 2; A is selected from the groupconsisting of

or a pharmaceutically acceptable salt, stereoisomer, or N-oxide thereof.14. The compound of claim 13 wherein m is selected from the groupconsisting of 0 and 1; R_(2a), R_(2b), and R_(2c), are independentlyselected from the group consisting of hydrogen, chloro, fluoro, methyl,trifluoromethyl, cyano, methoxy, and N(CH₃)₂; n is selected from thegroup consisting of 0 and 1; and R₃ is independently selected from thegroup consisting of fluoro, chloro, methyl, trifluoromethyl, cyano,methoxy, and NR₄R_(4′) where R₄ and R_(4′) are methyl, or alternativelytogether with the said N form the ring aziridin-1-yl or morpholino. 15.The method of claim 9 wherein the disorder is selected from asthma,chronic obstructive pulmonary disease (COPD) and cystic fibrosis.